Targeted liposome gene delivery

ABSTRACT

Targeted ligand-liposome-therapeutic molecule complexes (vectors) for the systemic delivery of the therapeutic molecule to various target cell types including cancer cells such as squamous cell carcinoma of the head and neck, breast and prostate tumors. The preferred ligands, folate and transferrin, target the liposome complex and facilitate transient gene transfection. The systemic delivery of complexes containing DNA encoding wild-type p53 to established mouse xenografts markedly sensitized the tumors to radiotherapy and chemotherapy. The combination of systemic p53 gene therapy and conventional radiotherapy or chemotherapy resulted in total tumor regression and long tern inhibition of recurrence. This cell-specific delivery system was also used in vivo to successfully deliver, via intravenous administration, small DNA molecules (oligonucleotides) resulting in chemosensitivity and xenograft growth inhibition. Other therapeutic molecules, including intact viruses, can be encapsulated in a complex and targeted in accordance with the invention.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is related to provisional applications serial Nos.60/066,188, filed Nov. 19, 1997, and 60/083,175, filed Apr. 27, 1998.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates generally to the systemic delivery of atherapeutic molecule via a liposome complex that is targeted to apre-selected cell type. More specifically, the invention providescompositions and methods for cell-targeted gene transfer and genetherapy for human cancers whereby a therapeutic molecule is delivered tothe targeted cancer cell via a ligand/liposome complex. Treatment ofcell proliferative disease (e.g. cancer) results in substantialimprovement of the efficacy of radiation and chemotherapeuticinterventions.

2. Description of Related Art

The ideal therapeutic for cancer would be one that selectively targets acellular pathway responsible for the tumor phenotype and would benontoxic to normal cells. To date, the ideal therapeutic remains justthat—an ideal. While cancer treatments involving gene therapy andanti-sense molecules have substantial promise, there are many issuesthat need to be addressed before this promise can be realized. Perhapsforemost among the issues associated with macromolecular treatments forcancer and other diseases is the efficient delivery of the therapeuticmolecule(s) to the site(s) in the body where they are needed.

A variety of nucleic acid delivery systems (“vectors”) to treat cancerhave been evaluated by others, including viruses and liposomes. Theideal vector for human cancer gene therapy would be one that could besystemically administered and then specifically and efficiently targettumor cells wherever they occur in the body. Viral vector-directedmethods show high gene transfer efficiency but are deficient in severalareas. The limitations of a viral approach are related to their lack oftargeting and to the presence of residual viral elements that can beimmunogenic, cytopathic, or recombinogenic.

A major deficiency of viral vectors is the lack of cancer cellspecificity. Absent tumor targeting capability, viral vectors arelimited in use to direct, local delivery that does not have thecapability to reach metastatic disease—the ultimate cause of death forthe majority of cancer patients.

The high titers achievable and the cell tropism that makes virusesattractive as gene therapy and gene transfer delivery vectors presentsome of their greatest deficiencies. Although the preparation of novelviruses with new targets for infection has been described in theliterature, these vectors are problematic due to the need for growingvirus to high titer. Consequently, a substantial amount of attention hasbeen directed to non-viral vectors for the delivery of moleculartherapeutics, including use in gene transfer and gene therapy.

Progress has been made toward developing non-viral, pharmaceuticalformulations of genes for in vivo human therapy, particularly cationicliposome-mediated gene transfer systems. Cationic liposomes are composedof positively charged lipid bilayers and can be complexed to negativelycharged, naked DNA by simple mixing of lipids and DNA such that theresulting complex has a net positive charge. The complex is easily boundand taken up by cells, with a relatively high transfection efficiency.Features of cationic liposomes that make them versatile and attractivefor DNA delivery include: simplicity of preparation, the ability tocomplex large amounts of DNA, versatility in use with any type and sizeof DNA or RNA, the ability to transfect many different types of cells(including non-dividing cells) and lack of immunogenicity orbiohazardous activity. The liposome approach offers a number ofadvantages over viral methodologies for gene delivery. Mostsignificantly, since liposomes are not infectious agents capable ofself-replication, they pose no risk of evolving into new classes ofinfectious human pathogens. Further, cationic liposomes have been shownto be safe and somewhat efficient for in vivo gene delivery. Sinceliposomes are not infectious agents, they can be formulated by simplemixing. Further, cationic liposomes have been shown to be safe andsomewhat efficient for in vivo gene delivery. Clinical trials are nowunderway using cationic liposomes for gene delivery, and liposomes fordelivery of small molecule therapeutics (e.g., chemotherapeutic andantifungal agents) are already on the market.

One disadvantage of cationic liposomes is that they lack tumorspecificity and have relatively low transfection efficiencies ascompared to viral vectors. However, targeting cancer cells via liposomescan be achieved by modifying the liposomes so that they bear a ligandrecognized by a cell surface receptor. Receptor-mediated endocytosisrepresents a highly efficient internalization pathway in eukaryoticcells. The presence of a ligand on a liposome facilitates the entry ofDNA into cells through initial binding of ligand by its receptor on thecell surface followed by internalization of the bound complex. Onceinternalized, sufficient DNA can escape the endocytic pathway to beexpressed in the cell nucleus.

There now exists a substantial knowledge base regarding the moleculesthat reside on the exterior surfaces of cancer cells. Surface moleculescan be used to selectively target liposomes to tumor cells, because themolecules that are found upon the exterior of tumor cells differ fromthose on normal cells. For example, if a liposome has the proteintransferrin (Tf) on its surface, it can target cancer cells that havehigh levels of the transferrin receptor.

A variety of ligands have been examined for their liposome-targetingability, including folic acid (folate), a vitamin necessary for DNAsynthesis, and transferrin. Both the folate receptor and transferrinreceptor levels are found to be elevated in various types of cancercells including ovarian, oral, breast, prostate and colon. The presenceof such receptors can correlate with the aggressive or proliferativestatus of tumor cells. The folate receptor has also been shown torecycle during the internalization of folate in rapidly dividing cellssuch as cancer cells. Moreover, the transferrin and folate-conjugatedmacromolecules and liposomes have been shown to be taken up specificallyby receptor-bearing tumor cells by receptor mediated endocytosis. Thusthe folate and transferrin receptors are considered to be useful asprognostic tumor markers for cancer and as potential targets for drugdelivery in the therapy of malignant cell growth.

Failure to respond to radiotherapy and chemotherapy represents an unmetmedical need in the treatment of many types of cancer. Often, whencancer recurs, the tumors have acquired increased resistance toradiation or to chemotherapeutic agents. The incorporation into cancertherapies of a new component which results in sensitization to thesetherapies would have immense clinical relevance. One way in which suchchemo/radio sensitization could be achieved is via targeted genetherapy.

An important role for p53 in the control of cellular proliferation bythe regulation of cell cycle events and induction of programmed celldeath (apoptosis) has been established. Since it appears that mostanti-cancer agents work by inducing apoptosis, inhibition of, or changesin, this pathway may lead to failure of therapeutic regimens. A directlink, therefore, has been suggested between abnormalities in p53 andresistance to cytotoxic cancer treatments (both chemo- andradiotherapy). It has also been suggested that the loss of p53 functionmay contribute to the cross-resistance to anti-cancer agents observed insome tumor cells. Various groups have established a positive correlationbetween the presence of mutant p53 and chemoresistance in mousefibrosarcomas and in primary tumor cultures from breast carcinomas,human gastric and esophageal carcinomas, as well as B-cell chroniclymphoblastic leukemia. In addition, chemosensitivity via apoptosisreportedly was restored by expression of wtp53 in non-small cell lungcarcinoma mouse xenografts carrying mutant p53.

A role for the tumor suppressor gene p53 in many critical cellularpathways, particularly in the cellular response to DNA damage, has beenestablished. These pathways not only include gene transcription, DNArepair, genomic stability, chromosomal segregation and senescence, butalso regulation of cell cycle events and the modulation of programmedcell death (apoptosis). For its role in monitoring DNA damage, p53 hasbeen christened “guardian of the genome.” Cancer cells are characterizedby genetic instability, and mutations in p53 have been found to occurwith extremely high frequency in almost all types of human cancers.Indeed, quantitative or qualitative alterations in the p53 gene aresuggested to play a role in over half of all human malignancies. Thepresence of p53 mutations in the most common types of human tumors hasbeen found to be associated with poor clinical prognosis. Moreover,mutant (mt) p53 is rarely found in some of the more curable forms ofcancer e.g., Wilms's tumor, retinoblastoma, testicular cancer,neuroblastoma and acute lymphoblastic leukemia.

Numerous studies have reported that the expression of wt p53 hassuppressed, both in vitro and in mouse xenograft models, the growth ofvarious malignancies, e.g., prostate, head and neck, colon, cervical andlung tumor cells. It has also been reported that a p53-liposome complexpartially inhibited the growth of human glioblastoma and human breastcancer xenografts in mice. In addition, Seung et al. usedliposome-mediated intratumoral introduction of a radiation-inducibleconstruct expressing TNF-α to inhibit growth of a murine fibrosarcomaxenograft after exposure to ionizing radiation. However, p53 expressionalone, while being able to inhibit tumor growth partially, has not beenable to eliminate established tumors in the long-term.

The normal development of mice lacking wtp53 and the observations of apost-irradiation G₁ block in p53-expressing cells suggests that wt p53functions in the regulation of the cell after DNA damage or stressrather than during proliferation and development. Since it appears thatmany conventional anti-cancer therapies (chemotherapeutics andradiation) induce DNA damage and appear to work by inducing apoptosis,alterations in the p53 pathway could conceivably lead to failure oftherapeutic regimens.

Lack of wt p53 function has also been associated with an increase inradiation resistance. The presence of mt p53 and the consequent absenceof a G₁ block have also been found to correlate with increased radiationresistance in some human tumors and cell lines. These include humantumor cell lines representative of head and neck, lymphoma, bladder,breast, thyroid, ovary and brain cancer.

Based on these considerations, gene therapy to restore wtp53 function intumor cells should re-establish the p53-dependent cell cycle checkpointsand the apoptotic pathway thus leading to the reversal of thechemo-/radio-resistant phenotypes. Consistent with this model,chemosensitivity, along with apoptosis, was restored by expression ofwtp53 in non-small cell lung carcinoma mouse xenografts carrying mtp53.Chemosensitivity of xenografts involving the p53-null lung tumor cellline H1299 and T98G glioblastoma cells and sensitivity of WiDr coloncancer xenografts to cisplatin has been demonstrated. Increased cellkilling by doxorubicin or mitomycin C was also shown in SK-Br-3 breasttumor cells by adenoviral transduction of wtp53. However, someconflicting reports indicate that the relationship between p53expression and chemoresistance may have a tissue or cell type-specificcomponent. The transfection of wtp53 by an adenoviral vector has alsobeen shown to sensitize ovarian and colo-rectal tumor cells to radiationIt has also been reported that adenoviral-mediated wtp53 delivery didrestore functional apoptosis in a radiation-resistant squamous cellcarcinoma of the head and neck (SCCHN) tumor line resulting inradiosensitization of these cells in vitro. More significantly, thecombination of intratumorally injected adeno-wtp53 and radiation led tocomplete and long-term tumor regression of established SCCHN xenografttumors.

The current invention departs from the conventional use of viral vectorsfor the delivery of therapeutic molecules for gene therapy, for exampleas disclosed by Roth et al. (U.S. Pat. No. 5,747,469). These currentlyused vehicles only have the limited capability of local delivery. Theirsuitability for intratumoral delivery has been shown not only to beinadequate in reaching all of the cells within the primary tumor mass,but also incapable of reaching sites of metastatic disease.

SUMMARY OF THE INVENTION

In one aspect, the invention provides cell-targetingligand/liposome/therapeutic molecule complexes for the in vitro or invivo delivery of therapeutic molecules to targeted cell types. Thecomplexes are useful as delivery vehicles (vectors) for delivering atherapeutic molecule to the target cells. The complexes are useful asvectors for carrying out gene transfer and gene therapy when thetherapeutic molecule is, for example, a nucleic acid encoding atherapeutic protein. Specific embodiments relate to folate andtransferrin-targeted cationic liposomes for the delivery of atherapeutic molecule to animal (including human) cancer cells thatcontain folate or transferrin receptors.

In another aspect, the invention provides pharmaceutical compositionscomprising a cell-targeting ligand/liposome/therapeutic molecule complexin a pharmaceutically compatible vehicle or carrier. The compositionsare formulated for, preferably, intravenous administration to a humanpatient to be benefitted by the effective delivery of the therapeuticmolecule. The complexes are appropriately sized so that they aredistributed throughout the body following i.v. administration.

In another aspect, the invention relates to therapeutic methodscomprising the administration to a warm-blooded animal (includinghumans) in need thereof, of a therapeutically effective amount of apharmaceutical composition comprising a ligand/liposome/therapeuticmolecule complex in a pharmaceutically acceptable vehicle. As set forthin detail herein, human cancer treatment via the systemic (e.g. i.v.)administration of a complex comprising a ligand-targeted liposomecomplex containing a nucleic acid encoding wt p53 is an importantembodiment of this aspect of the invention.

Human gene therapy via the systemic administration of pharmaceuticalcompositions containing targeted liposome/nucleic acid complexes,wherein the nucleic acid comprises a therapeutic gene under the controlof an appropriate regulatory sequence, form important examples of theinvention. Gene therapy for many forms of human cancers is accomplishedby the systemic delivery of folate or transferrin-targeted cationicliposomes containing a nucleic acid encoding wt p53. The data presentedherein demonstrates the superior ability of such complexes tospecifically target and sensitize tumor cells (due to expression of thewt p53 gene), both primary and metastatic tumors, to radiation and/orchemotherapy both in vitro and in vivo.

Yet another aspect of the invention relates to improvements to thepreparation of liposomes, especially ligand-targeted cationic liposomes,whereby liposomes of relatively small, consistent diameters areprovided. The consistent, small-diameter liposomes, followingintravenous administration, exhibit the ability to circulate in thebloodstream and target both primary tumors and metastases.

The present invention addresses the need to deliver therapeuticmolecules systemically with a high degree of target cell specificity andhigh efficiency. When systemically administered, the complexes of thepresent invention are capable of reaching, and specifically targeting,metastatic as well as primary disease, when the target cells are humancancer cells. As a result of delivery of the normal, wild-type versionof the tumor suppressor gene p53 by means of this system, the inventorsdemonstrated that the tumors are sensitized to radiation therapy and/orchemotherapy. The high transfection efficiency of this system results insuch a high degree of sensitization that not only is there growthinhibition of the cancer but pre-existing tumors and metastases arecompletely eliminated for an extended period of time. In some instancesthis period of time is such that the disease may be considered to becured.

The exceptional efficacy of this system is due in part to theligand-targeting of the liposome-therapeutic molecule complex. Moreover,the specific cationic and neutral lipids that comprise the liposomecomplex, as well as the ratio of each, have been varied and optimized sothat the efficiency of uptake of the therapeutic molecule would be idealfor the specific target cell type. The ratio of liposome to therapeuticmolecule was also optimized for target cell type. This optimization ofthe liposome-therapeutic molecule complex, in combination with theaddition of a targeting ligand, yields substantially improved efficacywhen administered in conjunction with radiation or chemotherapies. Thoseskilled in the art will be able to optimize the complexes for deliveringa variety of therapeutic molecules to a variety of cell types.

An important feature of the invention resides in the ability to deliverthe therapeutic molecule to the target cell through intravenous,systemic administration. The ability to efficiently target and transfectspecific cells following intravenous administration is accomplished bythe disclosed combination of selecting an appropriate targeting ligandand optimizing the ratio of cationic to neutral lipid in the liposome.In the case where tumor cells are the target cells, systemic delivery ofthe ligand-liposome-therapeutic molecule complex allows the efficientand specific delivery of the therapeutic molecule to metastases as wellas primary tumor.

The invention is not limited to the use of any specific targetingligand. The ligand can be any ligand for which a receptor isdifferentially expressed on the target cell. The presently preferredligands are folic acid (esterified to a lipid of the liposome) andtransferrin, and each of these ligands possesses advantageousproperties.

Liposome complexes are capable of penetrating only approximately twentylayers of cells surrounding the blood vessels in a tumor. It has beenpostulated that wtp53 gene therapy controls cell growth partiallythrough a “bystander” effect, which may be related to the induction ofapoptosis by wtp53. This “bystander effect” may account for theeffectiveness of the in vivo studies reported herein and may be acontributory factor to the effectiveness of the combination therapy.However, relatively little is known at this time concerning themechanism and pathway involved in this process for p53. It has beenspeculated that some as yet unknown apoptotic signal may be containedwithin the vesicles, which result from apoptosis, and which neighboringcells ultimately phagocytize. Alternatively, this apoptotic signal maybe transferred through gap junctions, as is believed to be the case forphosphorylated gancyclovir with the HSV-TK gene. Induction ofanti-angiogenic factors may also contribute to the bystander effect.

It has recently been reported that a non-targeted p53-liposome complexpartially inhibited the growth of human glioblastoma xenografts in vivo.In addition, Seung et al. (Cancer Res. 55, 5561-5565 (1995) usedcommercial non-targeted liposome (Lipofectin) mediated intratumoralintroduction of a radiation inducible construct containing TNF-α topartially inhibit xenograft growth of a murine fibrosarcoma afterexposure to 40 Gy ionizing radiation. Xu et al. (Human Gene Therapy 8,177-175 (1997)) showed that introduction of 16 μg of p53 DNA in anon-targeted liposome complex was able to partially inhibit the growthof breast cancer xenograft mouse tumors. However, the ligand-directedliposome-p53 complexes of the present invention provide the capacity fortarget cell specificity, and high transfection efficiency, coupled withsystemic administration. The studies reported here are the first toemploy such a delivery system in combination with conventional radiationand chemotherapeutic treatment for tumors. While p53 gene therapy alonemay not be sufficient to completely eliminate tumors long term, thepresently-described combination of liposome-mediated p53 gene therapyand conventional (radiation and/or chemotherapy) therapy was able toachieve not only growth inhibition, but tumor regression, demonstratinga synergistic effect.

The in vivo studies described herein demonstrate that the combination ofsystemic LipF-p53 or LipT-p53 gene therapy and conventional radiotherapyand/or chemotherapy was markedly more effective than either treatmentalone. In the clinical setting, radiation doses of 65 to 75 Gy for grosstumor and 45 to 50 Gy for microscopic disease are commonly employed inthe treatment of head and neck cancer. Given the known, adverse sideeffects associated with high doses of radiation or chemotherapy,sensitization of tumors so as to permit a lowered effective dose of theconventional treatment would be of immense clinical benefit.Furthermore, in the case of radiation, systemic restoration of wtp53function, resulting in a decrease in the radiation treatment dose foundto be effective, would permit further therapeutic intervention fortumors which did reoccur.

In reports using xenograft tumors derived from SCCHN cell linescontaining either wtp53 or mtp53 it was noted that introduction ofwtp53, via intratumoral administration of an adenoviral vector, was ableto inhibit the development of, and induce apoptosis in, these xenografttumors independent of their endogenous p53 status. Similarly,liposome-mediated introduction of wtp53 into both glioblastoma (RT-2)and breast cancer (MCF-7) xenografts, which have endogenous wtp53, wasable to partially inhibit the growth of these tumors. These studiesindicate the broad potential of wtp53 gene therapy, irrespective of p53gene status.

The research underlying the present invention demonstrates that theligand-cationic liposome-therapeutic molecule complex system can deliverthe p53 gene in vivo selectively to tumors of various types, sensitizingthem to radiation and chemotherapy. Consequently, systemic wtp53 genetherapy, mediated by the tumor-targeting, relatively safe and efficientligand-targeted cationic liposome system, in combination withconventional radiotherapy or chemotherapy, may provide a more effectivetreatment modality not only for primary tumors, but also for thosecancers which fail initial therapy.

It also has been demonstrated that the targeted liposome delivery systemis capable of delivering small DNA molecules (e.g. antisenseoligonucleotides), as well as agents as large as intact viral particles.Delivery of these small (antisense) DNA molecules was also able tosensitize tumor cells to chemotherapeutic agents. Thus, the targetedliposomes of the present invention are widely applicable to the systemicdelivery of therapeutic agents.

The invention also relates to methods for preparingligand-liposome-therapeutic agent complexes. The method by which thecomplex is formed between the transferrin-liposome and viral particleprovides a large number of transferrin molecules upon the surface of thecomplex and thereby increases the stability of the complex as it travelsthrough the blood stream. Moreover, when the therapeutic molecule is aviral particle, the transferrin liposome may also serve to decrease theimmunogenicity of the virus by blocking viral antigens.

Using the present invention, the inventors have demonstrated aremarkable effect not only in controlling cell growth, in particulartumor cell growth, but also in effecting tumor remission long-term.Tumor cell formation and growth, also known as transformation, describesthe formation and proliferation of cells that have lost their ability tocontrol cell division, that is, they are cancerous. A number ofdifferent types of transformed cells can serve as targets for themethods and compositions of the present invention, such as: carcinomas,sarcomas, melanomas, and a wide variety of solid tumors and the like.Although any tissue having malignant cell growth may be a target, headand neck, breast, prostate, pancreatic, glioblastoma, cervical, lung,liposarcoma, rhabdomyosarcoma, choriocarcinoma, melanoma,retinoblastoma, ovarian, gastric and colorectal cancers are preferredtargets.

It is further contemplated that the invention can also be used to targetnon-tumor cells for delivery of a therapeutic molecule. While any normalcell can be a target, the preferred normal targets are dendritic cells,endothelial cells of the blood vessels, lung cells, breast cells, bonemarrow cells, and liver cells.

It is disclosed herein that, when delivered systemically, theligand-targeted, optimized cationic liposomal-therapeutic moleculecomplex was able to specifically target and markedly sensitize tumorcells to radiation and/or chemotherapy resulting in substantial growthinhibition and tumor regression. The ligand-targeted, optimized cationicliposomal-therapeutic molecule complex may be delivered via other routesof administration such as intratumoral, aerosol, percutaneous,endoscopic, topical, intralesional or subcutaneous administration.

The invention provides, in certain embodiments, methods and compositionsfor the highly target cell-specific and efficient delivery, via systemicadministration, of a ligand-targeted, liposomal-therapeutic moleculecomplex. Examples of therapeutic molecules include a gene, highmolecular weight DNA, plasmid DNA, an antisense oligonucleotide,peptides, ribozymes, peptide nucleic acids, a chemical agent such as achemotherapeutic molecule, or any large molecule including, but notlimited to, DNA, RNA, viral particles, growth factors cytokines,immunomodulating agents and other proteins, including proteins whichwhen expressed present an antigen which stimulates or suppresses theimmune system.

Recently, efficient methods for long term expression of gene therapyvectors have been described (Cooper, et al, 1997; Westphal et al., 1998;Calos, 1996 and 1998). These vectors can be useful for extending and/orincreasing the expression levels of the disclosed liposomal deliverysystem. Several autonomous and episomal vector systems are disclosed inU.S. Pat. Nos. 5,707,830 (Calos, M. P., 13 Jan. 1998); 5,674,703 (Woo,S., et al., 7 Oct. 1997) and 5,624,820 (Cooper, M. J., 29 Apr. 1997)each of which is incorporated by reference herein. Calos relates toEpstein Barr virus-based episomal expression vectors useful inautonomous replication in mammalian cells. Woo et al. relates topapilloma virus-based episomal expression vectors for replication inanimal cells. Cooper et al. relates to vectors containing at least onepapovavirus origin of replication and a mutant form of papovavirus largeT antigen for long term episomal expression in human gene therapy.

When the therapeutic molecule is the p53 gene or an antisenseoligonucleotide, delivery via the complex of the invention results inthe sensitization of a cell or cells, such as a malignant cell or cells,to either radiation or a chemotherapeutic agent such that the cells arekilled via the combination therapy. Malignant cells are defined as cellsthat have lost the ability to control the cell division cycle as leadsto a transformed or cancerous phenotype. In addition to malignant cells,cells that may be killed using the invention include e.g., undesirablebut benign cells, such as benign prostatic hyperplasia cells,over-active thyroid cells, lipoma cells, as well as cells relating toautoimmune diseases such as B cells that produce antibodies involved inarthritis, lupus, myasthenia gravis, squamous metaplasia, dysplasia andthe like.

The ligand-liposome-therapeutic molecule complex can be formulated understerile conditions within a reasonable time prior to administration. Ifthe therapeutic molecule is one which provides enhanced susceptibilityto another therapy (such as enhanced susceptibility of cancer cells tochemotherapy or radiation therapy), such other therapy may beadministered before or subsequent to the administration of the complex,for example within 12 hr to 7 days. A combination of therapies, such asboth chemotherapy and radiation therapy, may be employed in addition tothe administration of the complex.

The terms “contacted” or “exposed” when applied to a cell are usedherein to describe the process by which a therapeutic molecule isdelivered to a cell, or is placed in direct juxtaposition with thetarget cell, so that it can effectively interact with the cell to bringabout a desired benefit to the cell or the host animal.

Wherein the complexes of the invention are used as an element of acombination therapy for, for example, human cancer treatment, they maybe used in combination with a wide variety of therapies employed in thetreatment of human or animal cancer. Such therapies include theadministration of chemotherapeutic agents and radiation therapies suchas gamma-irradiation, X-rays, UV irradiation, microwaves, electronicemissions and the like. Chemotherapeutic agents such as doxorubicin,5-fluorouracil (5FU), cisplatin (CDDP), docetaxel, gemcitabine,pacletaxel, vinblastine, etoposide (VP-16), camptothecia, actinomycin-D,mitoxantrone and mitomycin C can be employed in combination therapiesaccording to the present invention.

A variety of different types of potentially therapeutic molecules can becomplexed with the cell-targeted ligand/liposome complexes of theinvention. These include, but are not limited to, high molecular weightDNA molecules (genes), plasmid DNA molecules, small oligonucleotides,RNA, ribozymes, peptides, immunomodulating agents, peptide nucleicacids, viral particles, chemical agents such as per se knownchemotherapeutic agents and drugs, growth factors, cytokines and otherproteins including those which, when expressed, present an antigen whichstimulates or suppresses the immune system. Therefore, in addition togene therapy, the present invention can be used for immunotherapy or forthe targeted delivery of drugs.

Diagnostic agents also can be delivered to targeted cells via thedisclosed complexes. Agents which can be detected in vivo afteradministration to a multi-cellular organism can be used. Exemplarydiagnostic agents include electron dense materials, magnetic resonanceimaging agents and radiopharmaceuticals. Radionuclides useful forimaging include radioisotopes of copper, gallium, indium, rhenium, andtechnetium, including isotopes ⁶⁴Cu, ⁶⁷Cu, ¹¹¹In, ^(99m)Tc, ⁶⁷Ga or⁶⁸Ga. Imaging agents disclosed by Low et al. (U.S. Pat. No. 5,688,488)are useful in the present invention, and that patent is incorporated byreference herein.

The ligand-liposome composition of the invention, which will becomplexed with the therapeutic molecule, can be comprised of a ligand, acationic lipid and a neutral or helper lipid, where the ratio ofcationic lipid to neutral lipid is about 1:(0.5-3), preferably 1:(1-2)(molar ratio). The ligand can be bound, e.g. via chemical coupling, tothe neutral lipid and mixed with cationic lipid and neutral lipid at amolar ratio of about (0.1-20):100, preferably (1-10):100, and morepreferably (2.5-5):100 (ligand-lipid:total lipids), respectively. Theligand-liposome will be mixed with DNA or other therapeutic molecules toform a complex. The DNA to lipid ratios will be in a range of about1:(0.1-50), preferably about 1:(1-24), and more preferably about1:(6-16) μg/nmol. For antisense oligonucleotides, the complex will beformed by mixing the liposome with oligonucleotides at a molar ratio ofabout (5-30):1 lipid:oligonucleotide, preferably about (10-25):1, andmost preferably about 10:1.

Alternatively, as in the case of transferrin, the ligand can simply bemixed with the cationic and neutral lipids. In this instance, thecationic liposomes will be prepared at a molar ratio of cationic lipidto neutral lipid of about 1:(0.5-3), preferably 1:(1-2). Transferrinwill be mixed with the cationic liposomes and then DNA or othertherapeutic molecules. The DNA/Lipid/Tf ratios will be in the range ofabout 1:(0.1-50):(0.1-100) μg/nmol/μg, preferably about 1:(5-24):(6-36),and more preferably about 1:(6-12):(8-15), respectively.

Another unique feature of the complexes according to the invention istheir evenly distributed relatively small size (mean diameter less thanabout 100 nm, preferably less than about 75 nm, and more preferablyabout 35-75 nm (50 nm average) diameter). To reach the target tumor, thecomplexes must be resistant to degratory agents encountered in vivo, andalso must be capable of passing through the blood vessel (capillary)walls and into the target tissue. The complexes of the present inventionexhibit high resistance to degradation by elements present in serum. Thepermeable size of the capillaries in tumors is usually 50-75 nm, and thecomplexes which are less than about 75 nm diameter can pass easilythrough the capillary wall to reach the target. Based upon transmissionelectron microscopy, it appears that a unique onion-like layeredstructure of the LipF-DNA and LipT-DNA complex plays an important rolein the small size and, consequently, high transfection efficiency of thecomplex of the invention observed in vitro and, in particular, in vivo.

The ligand can be any molecule that will bind to the surface of thetarget cell, but preferentially to a receptor that is differentiallyexpressed on the target cell. Two particularly preferred ligands arefolate and transferrin. The cationic lipid can be any suitable cationiclipid, but dioleoyltrimethylammonium-propane (DOTAP) and DDAB arepreferred. The neutral lipid can be any neutral lipid, and preferredneutral lipids are dioleoylphosphatidylethanolamine (DOPE) andcholesterol.

A number of in vitro parameters may be used to determine the targetingand delivery efficiency of the composition so that particular complexescan be optimized to deliver a desired therapeutic molecule to theselected target cell type. These parameters include, for example, theexpression of marker genes such as the β-galactosidase or luciferasegenes, immunohistochemical staining of target cells for the deliveredprotein, Western blot analysis of the expression of the protein productof the delivered gene, down-modulation of the target gene due to adelivered anti-sense or other inhibitory oligonucleotide, as well asincreased sensitization of the target cells to radiation and/orchemotherapeutic agents.

In a preferred embodiment, it is contemplated that the p53 expressionregion will be positioned under the control of a strong constitutivepromoter such as an RSV or a CMV promoter. Currently, a particularlypreferred promoter is the cytomegalovirus (CMV) promoter.

The methods and compositions of the present invention are suitable fortargeting a specific cell or cells in vitro or in vivo. When the targetcells are located within a warm-blooded animal, e.g. head and neck,breast, prostate, pancreatic or glioblastoma cells, theligand-liposome-therapeutic molecule complex will be administered to theanimal in a pharmacologically acceptable form. A “pharmacologicallyacceptable form”, as used herein refers to both the formulation of theligand-liposome-therapeutic molecule complex that may be administered toan animal, and also the form of contacting an animal with radiation,i.e. the manner in which an area of the animals body is irradiated, e.g.with gamma-irradiation, X-rays, UV-irradiation, microwaves, electronicemissions and the like. The use of DNA damaging radiation and waves isknown to those skilled in the art of radiation therapy.

The present invention also provides improved methods for treatingcancer, both primary and metastatic, that, generally, compriseadministering to an animal or human patient in need thereof atherapeutically effective combination of a ligand-liposome-therapeuticmolecule (e.g. p53 gene) complex, and a therapy such as radiation orchemotherapy.

The complex will generally be administered to the animal, usuallysystemically, in the form of a pharmaceutically acceptable composition.In the preferred embodiment, the composition would be deliveredsystemically through an intravenous route. However, other routes ofadministration such as aerosol, intratumoral, intralesional,percutaneous, endoscopic, topical or subcutaneous may be employed.

The high degree of tumor cell specificity and tumor targeting ability ofthe invention was demonstrated by the expression of a reporter geneafter systemic delivery by the folate/transferrin-liposome-β-Gal genecomplex. β-galactosidase expression was evident in up to 70% of thexenografts of various human tumor cells, including JSQ-3, DU145 andMDA-MB-435, while normal tissues and organs, including the highlyproliferative gut and bone marrow, showed no evidence of transfection.The highly efficient tumor targeting ability of the invention was alsoevident in these experiments where metastases, and even micro-metastasesas small as a few cells, were found to have been specificallytransfected after systemic delivery of the complex.

The surprising success of the present invention is evidenced by thefinding that systemic delivery of either folate-liposome-wtp53 gene ortransferrin-liposome-wtp53 gene, in combination with either radiation orchemotherapy, yielded profound results in studies using a nude mousemodel. The high efficiency of this system results in such a high degreeof sensitization of JSQ-3 and DU145 human xenograft tumors to radiationthat not only is there growth inhibition of the cancer but, in someexperiments, the pre-existing tumors and metastases were completelyeliminated for an extended period of time. In some instances this periodof time (more than one year disease-free) is such that the disease maybe considered to be cured. Human breast cancer MDA-MB-435 and humanpancreatic cancer PANC I nude mouse xenograft tumors were also shown tobe highly sensitized by the systemic administration of eitherfolate-liposome-wtp53 or transferrin-liposome-wtp53 to chemotherapeuticagents including doxorubicin, cisplatin, docetaxel or gemcitabine.

As used herein, the term “transfection” is used to describe the targeteddelivery of a therapeutic molecule to eukaryotic cells using theligand-liposome complex of the invention and entry of the therapeuticmolecule into the cell by various methods, such as receptor mediatedendocytosis. The target cell may be preferentially selected by theligand of the complex such that the ligand will bind to a receptor thatis differentially expressed on the surface of the target cell.

Preferred pharmaceutical compositions of the invention are those thatinclude, within a pharmacologically acceptable solution or buffer, acomplex consisting of a ligand, a cationic-neutral liposome and atherapeutic molecule.

Still further embodiments of the present invention are kits for use inthe systemic delivery of a therapeutic molecule by the ligand-liposomecomplex, as may be formulated into therapeutic compositions for systemicadministration. The kits of the invention will generally comprise, inseparate, suitable containers, a pharmaceutical formulation of theligand, of the liposome and of the therapeutic molecule. In thepreferred embodiment the ligand would be either folate or transferrin,the liposome would consist of a cationic and a neutral lipid and thetherapeutic molecule would be either a construct carrying wtp53 undercontrol of the CMV promoter, or an antisense oligonucleotide. The threecomponents can be mixed under sterile conditions and administered to thepatient within a reasonable time frame, generally from 30 min to 24hours, after preparation.

The components of the kit are preferably provided as. solutions or asdried powders. Components provided in solution form preferably areformulated in sterile water-for-injection, along with appropriatebuffer(s), osmolarity control agents, antibiotics, etc. Componentsprovided as dry powders can be reconstituted by the addition of asuitable solvent such as sterile water-for-injection.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention uses systemic administration of a ligand/cationicliposomal delivery complex for tumor-targeted delivery of a therapeuticmolecule via receptor-mediated endocytosis. In one of the preferredembodiments, the ligand-targeted liposomes are used to deliver atherapeutic molecule comprising a gene encoding wild-type (wt) p53. Thetherapeutic gene is targeted and effectively delivered to tumor cells,resulting in the restoration of the normal p53 gene function that manytumors lack. This restoration has a profound effect on the ability totreat the tumors. In another preferred embodiment, the therapeuticmolecules being delivered are antisense oligonulceotides directedagainst genes in the cell growth pathway. Down-modulation of these genesresults in sensitization of the tumor cells and xenografts to radiationand chemotherapeutic agents. In yet another embodiment, the “therapeuticmolecule” is an intact viral vector (e.g. an adenoviral or retroviralparticle containing a therapeutic nucleic acid) which is delivered tothe targeted cell via the ligand/liposome complex.

In another aspect, the invention provides compositions and methods foraccomplishing gene therapy to restore wtp53 function in tumor cells,leading to the reversal of chemo-/radio-resistant phenotypes andconsequently improving the ability to treat the tumor via chemo- and/orradiation therapy.

The present invention provides a new and improved method foraccomplishing cancer gene therapy by providing a systemic deliverysystem (“complex”) that specifically targets tumor cells, includingmetastases, and results in a more effective cancer treatment modality.This method uses a ligand-directed cationic liposome system to deliver atherapeutic molecule to the tumor cells. In one of the preferredembodiments, this therapeutic molecule is wtp53. The inclusion of acell-targeting ligand (e.g. the folate or transferrin ligand) in theliposome-DNA complex takes advantage of the tumor-targeting facet andreceptor-mediated endocytosis associated with the ligand to introducewtp53 efficiently and specifically to the tumor cells in vivo as well asin vitro. The consequence of this restoration of wtp53 function is anincrease in sensitization to conventional radiation and chemo-therapies,thereby increasing their efficacy and/or reducing the total dosethereof.

The exemplified liposome compositions are based upon the cationic lipidDOTAP and fusogenic neutral lipid DOPE conjugated (e.g. esterified) toeither folic acid (to provide a folate ligand thereon) or simply mixedwith iron-saturated transferrin. The ratio of lipids themselves, as wellas the lipid:DNA ratio, will be optimized for in vivo delivery, as wellas for different tumor cell types, e.g. adenocarcinoma vs. squamous cellcarcinoma. In vitro studies demonstrated that the addition of the ligandsubstantially increased the transfection efficiency for tumor cells whencompared to the liposome alone, even in the presence of high levels ofserum. Transfection of wtp53 by this method resulted in substantialradiosensitization of a previously radiation resistant SCCHN cell linein vitro.

The in vivo tumor targeting capability of this system was assessed usingthe β-galactosidase reporter gene in three different types ofcancer—SCCHN, breast cancer and prostate cancer. These studiesdemonstrated that after intravenous administration of the complexes,only the tumors were transfected, with an efficiency between 50 and 70%,while normal organs and tissues, including the highly proliferative bonemarrow and intestinal crypt cells, showed no signs of reporter geneexpression. Some ligand-liposome-DNA complex was evident in macrophages.Very significantly, even micro-metastases in the lung, spleen and lymphnodes showed evidence of highly efficient and specific transfection.

When the systemically delivered ligand-liposome wtp53 complex wasadministered to mice bearing radiation resistant human SCCHN xenografts,and followed with radiation therapy, the tumors completely regressed.Histological examination of the area of the former tumor showed onlynormal and scar tissue remaining, with no evidence of live tumor cells.This was in contrast to the tumors from animals treated only with theligand-liposome-p53 complex or only with radiation. In these animalssome cell death was evident. However, nests of live tumor cellsremained, resulting in the regrowth of the tumors in these animals.Strikingly, no recurrence of the tumors was evident in the animalsreceiving the combination therapy, even one year after the end oftreatment. Similar results were observed in mice bearing human prostatetumor xenografts with radiation and chemotherapeutic agents, as well aswith human breast cancer and pancreatic cancer xenografts withchemotherapeutic agents. Consequently, this system is viewed asproviding a more effective form of cancer therapy.

Therefore, the present invention represents a significant improvementupon current experimental cancer therapies, such as local injection ofadenoviral vectors carrying a therapeutic molecule such as p53, whichare frequently incapable of administering a therapeutic molecule to theentire tumor tissue (primary tumor mass). Local delivery also lacks thecapability of reaching distant metastases. The specific targetingability provided by the present invention is also advantageous since itreduces side effects that can be associated with wide spreadnon-specific uptake of the therapeutic molecule.

The uptake of the ligand-liposome-therapeutic molecule complex by thetarget cells will, when administered in conjunction with adjuvanttherapies, and when the target cells are cancer cells, not only decreasethe rate of proliferation of these cells but actually result inincreased tumor cell death and long-term tumor regression. The deliverysystem of the invention strongly portends a prolongation of patientsurvival.

Even though the invention has been described with a certain degree ofparticularity, it is evident that many alternatives, modifications, andvariations will be apparent to those skilled in the art in light of thepresent disclosure. Accordingly, it is intended that all suchalternatives, modifications, and variations which fall within the spiritand the scope of the invention be embraced by the defined claims.

BRIEF DESCRIPTION OF DRAWINGS

FIGS. 1A and 1B show the adenoviral shuttle plasmids pRSVp53, pRSVpRo,pCMVp53 and pCMVpRo that contain the sense and anti-sense cDNA of p53.

The following examples are included to demonstrate preferred embodimentsof the invention.

EXAMPLE 1 Construction of p53 Expression Vector

This example describes the construction of p53 expression vectors. Themethods used are those commonly known to those skilled in the art. Theinvention is not limited to any particular expression vector, however.

The adenoviral shuttle plasmids pRSVp53, pRSVpRo, pCMVp53 and pCMVpRothat contain the sense and anti-sense cDNA of p53 are shown in FIG. 1.These plasmids were constructed by cloning the 1.7 Kb XbaI p53 cDNAfragment into an adenoviral shuttle vector. Davidson, et al.,Experimental Neurobiology 125, 258-267 (1994) is incorporated byreference herein for the purpose of disclosing the preparation of suchshuttle vectors. Orientation was determined by restriction digest andconfirmed by DNA cycle sequencing. The plasmids were expanded in E. coliDH5α and purified by Qiagen Plasmid Mega/Giga Kits (Qiagen). Thepurified plasmids were quantified spectrophotometrically with A₂₆₀/A₂₈₀values approximately 1.90. Agarose gel (0.8%) electrophoresis confirmedthat more than 95% of plasmid DNA was supercoiled.

EXAMPLE 2 Synthesis of Ligand-liposome-DNA Complexes

This example describes one method suitable for the production of theligand-liposome-therapeutic molecule complex, where the therapeuticmolecule is plasmid DNA. 15 mmol dioleoylphosphatidylethanolamine (DOPE)in dry chloroform was reacted with 20 mmol N-hydroxysuccinimide ester offolic acid (see Lee, R. J., et al., J. Biol. Chem. 269, 3198-3204(1994), which is incorporated by reference herein for the purpose ofdisclosing such procedures) in the presence of 20 mmol triethylamine for4 hours at room temperature, then washed with PBS 3 times to obtainfolate-DOPE in chloroform. Thin-layer chromatography (chloroform:methanol: acetic acid, 80:20:5) revealed that more than 95% of DOPE(Rf=0.65-0.70) was converted to folate-DOPE (Rf=0.90-0.95). LipF(A) wasprepared as follows: a chloroform solution of 5 μmoldioleoyltrimethylammonium-propane (DOTAP), 5 μmol DOPE and 0.1 μmolfolate-DOPE were mixed together in a round-bottom flask, and thechloroform evaporated under reduced pressure. 10 ml sterile water wasadded to the flask to suspend the lipids, then sonicated for 10 min in abath-type sonicator at 4° C. The final concentration of LipF(A) was 1nmol/μl total lipids. The LipF(A)-DNA complex for in vitro use wasprepared by mixing equal volumes of LipF(A) and DNA in serum-freeRPMI-1640 folate free medium (Life Technologies, Inc.) and incubating atroom temperature, with frequent rocking, for 15-30 minutes. DNAretardation assay showed that at the ratio of 1 μg DNA: 8-10 nmolLipF(A), almost all of the added DNA was complexed with lipids. For invivo experiments, plasmid DNA (diluted in HEPES buffer, pH 7.4 basedupon the total amount of DNA/mouse) was mixed with LipF(A)(in water) atthe ratio of 1 μg DNA/8-12 nmol lipids, and incubated for 15-30 minutesat room temperature with frequent rocking. A 50% dextrose solution wasadded to reach a final concentration of 5% dextrose, mixed by inversionand checked for signs of precipitation (the presence of particulatematter or cloudiness). In both cases, the LipF(A)-DNA complexes werefound to be stable for up to 24 hour at 4° C. in the dark, withoutsubstantial loss of transfection efficiency.

Cationic liposomes consisting of dioleoyl trimethylammonium propane(DOTAP) and dioleoyl phosphatidylethanolamine (DOPE) (Avanti PolarLipids, Inc., Alabaster, AL) were prepared as above. The finalconcentration of liposomes was 2 nmol/ul. Holo-transferrin (Tf,iron-saturated, Sigma) was dissolved in pure water at 5 mg/ml. TheTf-liposome-DNA complex for in vitro experiments was prepared asdescribed by Cheng, P. W., Human Gene Therapy 7, 275-282 (1996) (whichis incorporated herein by reference for the purpose of illustratingliposome preparation) with modifications. In brief, 12 nmol of liposomeswere added to 18 mg Tf in 100 μl serum-free EMEM and incubated for 5-15min at room temperature with frequent rocking. This solution was thenmixed with 1.2 μg plasmid DNA in 100 μl serum-free EMEM and incubatedfor 15-30 minutes at room temperature with frequent rocking. Theprepared Tf-liposome (designated LipT(A))-DNA complex was used for invitro cell transfection freshly within 1 hour of preparation, althoughit was found to be stable for at least 24 hours with the sametransfection efficiencies. Agarose gel electrophoresis was employed toassess the DNA retardation by LipT(A). Greater than 90% of the DNA wasfound to be complexed to the liposome. For in vivo studies, the liposomeand transferrin (in water) were mixed and incubated for 5-15 minutes atroom temperature with frequent rocking. This solution was then mixedwith DNA (in HERPES buffer pH=7.4) and incubated for 15-30 minutes atroom temperature with frequent rocking. A 50% dextrose solution wasadded to reach a final concentration of 5% dextrose, mixed by inversionand checked for signs of precipitation (the presence of particulatematter or cloudiness). In both cases, the LipT(A)-DNA complexes werefound to be relatively stable for up to 24 hours at 40° C. in the dark,without substantial loss of transfection efficiency.

EXAMPLE 3 Folate-Liposome Optimization by X-Gal Staining

This example describes the optimization of the folate cationic-liposome(LipF) complex of the invention for squamous cell carcinoma of the headand neck (SCCHN). To optimize the transfection efficiency for SCCHN cellline JSQ-3, the E. coli LacZ gene, driven by an SV40 promoter in plasmidpSVb, was employed as a reporter. Transfection efficiency was calculatedbased upon the percent of X-Gal stained cells. As shown in Table 1, thepresence of folate ligand in the complex substantially increased thereporter gene expression. The non-ligand linked cationic liposome(Lip(A)) gave a transfection efficiency of 10%-20% in JSQ-3, in vitro,while LipF(A) resulted in 60%-70% of the cells expressing theβ-galactosidase gene. The addition of 1 mM free folic acid to the cellsprior to transfection was able to block the folate receptors on thecells, thereby reducing the transfection efficiency to 20%, similar tothat observed with LipF(A). These results demonstrate that using folateas a ligand increases the transfection efficiency of cationic liposomes,and that this effect is mediated by the folate receptor. Based upon arecent report that X-gal staining may underestimate the extent ofβ-galactosidase gene expression by 20% or higher, it is conceivable thatthe transfection efficiency with the ligand-targeted liposome mayactually exceed the 70% stated above.

TABLE 1 In vitro transfection efficiencies for LipF(A) in JSQ-3 cells:*Transfected by Without Serum With Serum PSVb alone    0%    0%Lip(A)-pSVb <20% <10% LipF(A)-pSVb 60%-70% 40%-50% LipF(A)-pSVb + 1 mMFolate** 15%-20% 10%-20% *60% confluent JSQ-3 cells, cultured infolate-free medium in a 24-well plate were transfected for 5 hours with0.5 ml of transfection solution containing 1.2 μg of pSVb. After anadditional 2 days in culture, the cells were fixed and stained withX-gal. Transfection efficiency was calculated as percent of blue stainedcells. **Folate was added immediately before transfection.

EXAMPLE 4 Optimization of LipT(A) System by Luciferase Assay

This example describes the optimization of the transferrincationic-liposome [LipT] complex of the invention for squamous cellcarcinoma of the head and neck (SCCHN). The LipT(A) system was optimizedfor JSQ-3 transfection using the luciferase assays. The fireflyluciferase gene driven by cytomegalovirus (CMV) promoter in plasmidpCMVLuc was employed as the reporter gene (Promega). 5×10⁴ JSQ-3cells/well were plated in a 24-well plate. 24 hours later, the cellswere washed once with EMEM without serum, 0.3 ml EMEM without serum orantibiotics was added to each well. The freshly preparedTf-liposome-pCMVLuc (LipT(A)-Luc) complex containing different amountsof plasmid DNA up to 1.0 μg in 0.2 ml EMEM was added to the cells. Aftera 5-hour incubation at 37° C. and 5% CO₂, 0.5 ml EMEM supplemented with20% fetal bovine serum and 1 μg/ml hydrocortisone were added to eachwell. 24 hours later, the cells were washed once with PBS, lysed with100 μl/well 1X Reporter Lysis Buffer (Promega), and the expressedluciferase activities were measured with Luciferase Assay System(Promega) on a Luminometer. A recombinant firefly luciferase (Promega)standard was used during each measurement for converting the luminometerreadings of relative light unit (RLU) to the equivalent amount ofluciferase expressed. Protein concentration of cell lysate was measuredusing the Bio-Rad DC Protein Assay Kit (Bio-Rad Laboratories). Theresults were expressed as μg of luciferase equivalent per mg of totalprotein. JSQ-3 cells were transfected with LipT(A)-pCMVLuc (LipT(A)-Luc)at different DNA/Lipid ratios in the complex. Transferrin substantiallyenhanced the transfection efficiency of cationic liposomes. Underoptimal condition, i.e., DNA/Lipid/Tf ratio at 1 ug/10 nmol/12.5 ug,luciferase was expressed at 12.5±1.1 ug/mg total protein, or 1.25% totalprotein, 7- to 10-fold more than liposome alone without transferrin.

EXAMPLE 5 In Vitro Transfection of JSQ-3 Cells by LipT(A)-pSVb

This example uses a quantitative β-galactosidase colorimetric assay, asdescribed in Example 3, to demonstrate the increased transfectionefficiency of the transferrin-liposome complex of the invention.Purified β-galactosidase (Boehringer) was used as standard. The resultswere expressed as milliunits (mU) of β-galactosidase equivalent per mgof total protein. For histochemical studies of Tf-liposome-pSVbtransfection, 60% confluent JSQ-3 cells in a 24-well plate weretransfected for 5 hours with 1.2 μg of pSVb with or without LipT(A).After an additional 2 days in culture, the cells were fixed and stainedwith X-gal. Transfection efficiency was calculated as percentage ofblue-stained cells. In quantitative β-galactosidase assay, the JSQ-3cells transfected at the optimal condition, with 0.5 ug DNA/10⁵ cells ofLipT(A)-pSVb, expressed 15.04±0.60 mU/mg total protein ofβ-galactosidase without serum, and 10.95±0.15 mU/mg with serum. Inhistochemical studies, transfection with LipT(A)-pSVb resulted in70%-80% of the cells being transfected. The presence of serum duringtransfection slightly reduced transfection efficiency, but even withserum, 40-50% of the cells stained blue, while cationic liposome withoutligand gave only 10-20% efficiency. These results demonstrated thatusing Tf as a ligand substantially increased the transfection efficiencyof cationic liposomes, even in the presence of serum.

EXAMPLE 6 Selective Tumor and Metastases Targeting by theLigand-liposome Complex In Vivo

This example demonstrates the ability of the folate or transferrincomplexed liposome to selectively target tumor tissue in vivo.Xenografts were induced by the subcutaneous injection of JSQ-3,MDA-MB-435 or DU145 cells. 2.5×10⁶ (JSQ-3) or 5×10⁶ (Du145) cells wereinjected on the lower back above the tail of 4-6 week old female athymicnude (NCr nu-nu) mice. 1×10⁷ MDA-MB-435 cells were injectedsubcutaneously into the mammary fat pad of the mice. For the metastasesmodel, 1×10⁶ JSQ-3 or MDA-MB-435 cells were intravenously injected, viathe tail vein, into the animals. LipF(A)-pSVb or LipF(D)-pSVb wasprepared as described in Example 2. LipF-pSVb or pSVb plasmid alone (in5% dextrose) were injected intravenously via the tail vein, at 25 μg ofplasmid DNA/300 μl/animal. Two days and 10 days after DNA injection, thetumors as well as mouse organs were excised, cut into 1 mm sections,washed once with PBS, and fixed with 2% Formaldehyde-0.2% glutaraldehydefor 4 hours at room temperature. The fixed tumor sections were washed 4times, each for 1 hr, and stained with X-Gal solution plus 0.1% NP-40(pH 8.5) at 37° C. overnight The stained tumor sections were embeddedand sectioned using normal histological procedures and counter-stainedwith nuclear fast red. Four sections per tumor were examined to evaluatethe β-galactosidase gene expression, as indicated by the blue stainedcells.

LipF(A)-pSVb or pSVb alone was intravenously injected into nude micebearing JSQ-3 xenografts. Within 48 hours, the LipF(A)-pSVb injectedgroup showed reporter gene expression in the tumors with an in vivotransfection efficiency of approximately 40-50%. In contrast, with pSVbplasmid alone, less than 1% of the tumor cells stained for theβ-galactosidase reporter gene. Ten days after i.v. administration ofLipF(A)-pSVb, both the percentage and intensity of blue staining in thetumors were substantially reduced, indicating that the LipF(A)-mediatedsystemic transfection is transient. Vital organs in LipF(A)-pSVbinjected mice showed only macrophages such as Kupffer cells (liver) ordust cells (lung) staining blue, while the hepatocytes and lung alveolicells themselves remained unstained. The selectivity of tumor targetingwas also shown where the tumor was found invading muscle. TheLipF(A)-pSVb transfected only the tumor while the muscle cells remainedunstained. More significantly, the highly proliferating bone marrow andintestinal crypt cells were apparently not transfected. Both the cryptcells and the bone marrow showed little if any (<1%) evidence ofreporter gene staining. The lack of LipF(A)-pSVb transfection in thebone marrow and crypt cells demonstrates that targeting is not anonselective, cell proliferation effect, but appears to be targeting thetumor cells. This is further demonstrated by no staining being evidentin the endothelial cells of blood vessels, although they were exposed tothe highest concentration of the LipF(A)-pSVb complex as it travelsthrough the blood stream. In addition, no staining was evident in thelymphoblastic growth centers in the spleen even though the dendriticcells displayed B-galactosidase staining.

A major problem in cancer recurrence and treatment is metastases. Totest for the ability of the LipF(A) complex to target tumor cells apartfrom the subcutaneous xenograft, JSQ-3 cells were i.v. injected intonude mice. By two weeks after the injection, simulated metastases(islands of tumor cells in multiple organs) formed. Animals were theninjected intravenously with LipF-pSVb and the simulated metastasesexamined for β-galactosidase expression. Extensive X-gal staining wasseen in a metastasis found in a thoracic lymph node. In this section, ablood vessel (BV) was found surrounded by the metastatic tumor cells.Although the tumor cells exhibited strong X-gal staining 20-25 layersfrom the blood vessel, no reporter gene expression was evident in theendothelial cells of the blood vessel, even though they were exposed tothe highest concentration of the LipF(A)-pSVb complex as it traveledthrough the blood stream. These results confirmed the tumor-selectivenature of the LipF(A) complex and demonstrated that metastases as wellas primary tumors can be targeted via folate-containing liposomes.

To assess the breadth of applicability this folate-linked, liposomemediated delivery system to cancers other than SCCHN, experiments werealso performed with xenografts of other human tumor cell lines includinghuman breast carcinoma cell lines MDA-MB-435, Hs578T, and human prostatecancer cell line DU145, which also carry mt p53. Here too, a single i.v.injection of LipF(A)-pSVb demonstrated tumor selectivity. A high levelof β-galactosidase expression was seen in the MDA-MB-435 mammary fat padtumor while the adjacent normal muscle tissue remained unstained.Reporter gene expression was not detected in non-tumor tissues or normalorgans including intestinal crypt cells and hepatocytes, whilesubcutaneous mammary fad pad xenografts showed an average of 50-70% bluestaining. Two weeks after i.v. injection of MDA-MB-435 cells, theLipF(A)-pSvb was systemically delivered via a single tail veininjection. Even small simulated breast metastases in the lung displayeda high level of staining, and the adjacent normal lung tissue remainedcompletely unstained.

Mice bearing DU145 xenografts were given a single i.v. injection ofLipF(B)pSVb. Tumors in these mice also showed reporter gene expressionrepresenting an in vivo transfection efficiency of at least 40-50%, avalue about 50-fold higher than achieved with plasmid alone.

The transferrin-liposome, liposome-pSVb and pSVb DNA complexes wereprepared in sterile 5% dextrose instead of HBSS, at a ratio of 1 μgDNA/10 nmol liposome/12.5 μg transferrin. The nude mouse tumor model wasestablished by subcutaneous injection of JSQ-3 cells in the flank of 4-6weeks old female nude mice. 30 μg pSVb DNA complexed with Tf-liposome in300 ml volume were injected into each mouse via tail vein with 1 ccsyringe and a 30 G needle. In the control groups, liposome-pSVb or pSVbDNA without liposome were injected. At 2 days, the tumors in miceinjected with LipT(A)-pSVb showed reporter gene expression representingan in vivo transfection efficiency of approximately 20-40%. In contrast,with pSVb plasmid alone, without liposome, less than 1% of the tumorcells stained for reporter gene expression. Ten days after intravenousadministration of LipT(A)-pSVb, both the percentage of positive cellsand the intensity of blue staining in the tumors were substantiallyreduced, indicating that the LipT(A)-mediated systemic transfection wastransient. Vital organs in mice injected with LipT(A)-pSVb showedstaining of only macrophages (such as dust cells of the lung and Kupffercells of the liver), whereas the hepatocytes and lung alveolar cellsremained unstained. No staining was evident in the lymphoblastic growthcenters in the spleen although the dendritic cells displayed modeststaining. In summary, the histological staining indicated that deliveryof the reporter gene by LipT(A) was selective with the human xenograftbeing most heavily stained.

EXAMPLE 7 Expression of Exogenous Wild-type p53 Protein in LipF (A)-p53and LipT (A)-p53 Transfected JSQ-3 Cells

This example demonstrates the expression of an exogenous gene, in thisparticular example wild-type p53, in cells being contacted andtransfected by the delivery system of the invention. Having optimizedthe transfection efficiency both in vitro and in vivo, LipF(A) orLipT(A) was complexed to p53 expression plasmid pCMVp53, which contains1.7 Kb cDNA of wt human p53 LipF(A)-p53) or (LipT(A)-p53). For DNA-doseresponse of p53 gene expression, 2×10⁵ JSQ-3 cells were plated in eachwell of a 6-well plate. After 24 hours, cells were washed once with EMEMwithout serum and antibiotics, transfected with 1 ml transfectionsolution containing LipF(A)-p53 with increasing amounts (0.25-8 μg/10⁵cells) of pCMVp53 plasmid DNA complexed with LipF(A) or, as control,LipF(A)-pRo. Alternatively, the cells were transfected with LipT(A)-p53or LipT(A)-pRo containing up to 4 μg plasmid DNA/2×10⁵ cells at theratio of 1 μg DNA/10 nmol liposome/15 μg Tf in EMEM. Five hours aftertransfection, 1 ml EMEM supplemented with 20% FBS and 1 1 μg/mlhydrocortisone were added and cultured for another 48 hours Thetransfected cells were collected and lysed in RIPA buffer (Santa CruzBiotechnology, Inc) and Western blot analysis was performed with thepantropic anti-p53 monoclonal antibody Ab-2 (Santa Cruz Biotechnology,Inc.) using standard procedures familiar to those skilled in the art. 40μg of total protein was loaded per lane.

For a time-course of p53 gene expression, 2×10⁵ JSQ-3 cells weretransfected with 2 μg pCMVp53 or pCMVpRo complexed with LipT(A). Thecells were collected every 24 hours up to 5 days after transfection andused for Western blot analysis.

To investigate the radiation effect on p53 gene expression, JSQ-3 cellswere transfected with LipT(A)-p53 or LipT(A)-pRo (2 μg DNA/2×10⁵ cells)for 2 days, then trypsinized and irradiated at graded doses up to 6 Gyof ¹³⁷Cs g-rays in a J. L. Shepard and Associates Mark I irradiator. Theirradiated cells were replated and cultured for further 2 and 4 daysbefore collecting for Western blot analysis.

After transfection into JSQ-3 cells in vitro, western blot analysisdemonstrated that transfection with LipF(A)-p53 resulted in DNA-dose andtime dependent expression of exogenous wtp53. To serve as control,LipF(A) was also complexed to plasmid pCMVpRo which carries wt p53 inthe reverse orientation (LipF(A)-pRo). P53 expression in LipF(A)-pRotransfected JSQ-3 cells was the same as in the untransfected cells.Wtp53 expression was evident at 24 hr after LipF(A)-p53 transfection,peaking at 48 hr and becoming negligible by 120-hr post-transfection,again demonstrating the transient nature of this gene delivery system.This transitory expression is advantageous as it allows for repeatedinjections without accumulation of wtp53.

Western blot analysis was employed to demonstrate that theLipT(A)-transduced wtp53 was being expressed in JSQ-3 cells.Transfection with increased doses of p53-expression plasmid pCMVp53complexed with LipT(A) (LipT(A)-p53) resulted in a DNA-dose dependentwtp53 expression, while no exogenous p53 expression was evident in JSQ-3cells transfected with LipT(A)-pRo, which carries the wtp53 cDNA inreverse orientation under the CMV promoter. Wtp53 expression startedfrom 24 hr after LipT(A)-p53 transfection and peaked on the second day,then reduced. Only traces of exogenous p53 were left 5 days aftertransfection, indicating that LipT(A)-mediated wtp53 expression wastransient.

When the JSQ-3 cells were irradiated at 48 hr after LipT(A)-p53 orLipF(A)-p53 transfection (the peak of wtp53 expression), the exogenouswtp53 expression was substantially increased in accordance with thegamma-irradiation doses and was stable for up to 4 days, i. e., 6 daysafter transfection. These results demonstrated that gamma-irradiationcan enhance and/or stabilize the exogenous wtp53, suggesting that theexogenous p53 is behaving in a way analogous to normal, endogenouswtp53, which is known to be stabilized by radiation exposure.

EXAMPLE 8 Sensitization of JSQ-3 to Radiation In Vitro

The presence of a mutated form of p53 has been shown to correlate withincreased radiation resistance in some human tumors and cell lines (8,10). Therefore, this example examined the effect of replacement of wtp53by LipF(A)-mediated transfection on radiation survival. LipF(A)-mediatedp53 transfection was able to sensitize JSQ-3 cells to radiation in a DNAdose-dependent manner. At the optimal transfection conditions, i.e., 3μg plasmid DNA per 10⁵ cells, the D₁₀ value was substantially reducedfrom the highly resistant level found in the untransfected cells(6.65±0.43 Gy) to 4.33±0.06 Gy (p <0.01). This represents approximatelya 6-10 fold sensitization to radiation killing. A D₁₀ value of 4.33 Gy(4 μg/10⁵ cells) is similar to that of a radiosensitive human fibroblastcell line H500 (D₁₀=4.50±0.05 Gy) and in the range considered to beradiosensitive. Neither the pCMVp53 plasmid alone, nor the LipF(A)-pRo,had a substantial sensitizing effect, based upon the D₁₀ values (p>0.05)(FIG. 5). In terms of survival, this decrease of more than 2 Gyrepresents a dramatic increase in sensitivity to the killing effects ofionizing radiation. Clinically, this shift in sensitivity may render aresistant tumor curable by conventional radiation doses.

EXAMPLE 9 Apoptosis Induced by p53 Transfection and Gamma-irradiation

This example demonstrated that the reintroduction of wtp53 using theoptimized transferrin-liposome complex of the invention was able torestore a functional p53 dependent apoptotic pathway. JSQ-3 cells weretransfected with LipT(A)-p53 or LipT(A)-pRo (1 to 3 μg DNA/2×10⁵ cells)and both the attached and floating cells were collected every day for 3days for analysis of the percent of apoptotic cells. Forradiation-induced apoptosis, the cells were transfected for 2 days, thentrypsinized and irradiated as described above in Example 8. The replatedcells were collected 4 days later for analysis of the percent ofapoptotic cells. The collected cells were stained with the AnnexinV-FITC Kit (Trevigen, Inc., Gaithersburg, Md.) according to themanufacturer's protocol. Annexin V-FITC binds specifically tophosphatidylserine present on apoptotic cells. The stained cells wereanalyzed using a FACStar cytometer (Becton & Dickinson).

To examine the effect of wtp53 restoration on the induction ofapoptosis, JSQ-3 cells were transfected with LipT(A)-p53 or LipT(A)-pRo.A clear induction of apoptosis was observed in LipT(A)-mediated wtp53restoration, in a dose-dependent manner. The percentage of apoptoticcells peaked on the second day of transfection which correlated with thelevels of wtp53 expression in the cells as revealed by Western blot. Toexamine the effect of irradiation on the induction of apoptosis, thetransfected cells were treated with different doses ofgamma-irradiation. 2 to 4 days later, the cells were stained withAnnexin V-FITC and analyzed by flow cytometry using FACStar (Becton &Dickinson). Gamma-irradiation induced substantial increase in thepercent of apoptotic cells only in LipT(A)-p53 transfected cells, from18.7% (0 Gy) to 38.7% (4 Gy) and 46.4% (6 Gy) 4 days after irradiation.No increase was observed in the untransfected (UT) cells and LipT(A)alone or LipT(A)-pRo treated cells. The increase was radiationdose-dependent and correlated with the wtp53 expression levels found inthe Western blot data, demonstrating that the radiation enhancement ofapoptosis was proportional to the wtp53 level in cells. That is, themore wtp53 that was expressed, the more apoptosis was induced.

EXAMPLE 10 Sensitization of JSQ-3 Xenograft Tumors to Radiation by theSystemic Delivery of LipF(A)-p53

In this example, the use of the systemically deliveredfolate-liposome-therapeutic molecule as a method of cancer treatment wasdemonstrated. In this particular example, the therapeutic molecule isthe normal human wild-type p53 gene (pCMVp53).

Squamous cell carcinoma of the upper aerodigestive tract results insignificant morbidity and mortality despite recent improvements intherapy Patients who present with early-stage disease (stage I or II)are generally treated with either surgery or radiation therapy, whilethe more common patients with advanced disease (stage III or IV) aregenerally treated with surgery followed by radiation. Despite this, halfor more of patients treated for advanced stage disease will relapse atthe site of original disease or with distant metastases and ultimatelydie. Presumably, a significant portion of these clinical failures resultfrom radiation resistance in a subset of tumor cells. Therefore, thedevelopment of an effective method for sensitizing head and neck tumorsto radiotherapy should have a profound effect on the treatment of thisdisease.

Mutant (mt) forms of the tumor suppressor gene p53 have been associatedin a number of studies with poor clinical prognosis for various types ofmalignancy. P53 may also be involved in the development and progressionof squamous cell carcinoma of the head and neck (SCCHN). By using avariety of detection methods, abnormalities in the p53 gene and/or itsexpression have been identified in 33%-100% of SCCHN tissues. Thepresence of mt p53 may also be indicative in SCCHN of increasedfrequency and more rapid recurrence of the tumor. Wild-type (wt) p53 hasbeen shown to function in the regulation of the cell cycle after DNAdamage or stress rather than during normal proliferation anddevelopment. Since the presence of mt p53 has also been found tocorrelate with increased radiation resistance (RR) in some human tumorsand cell lines, and because a high percentage of head and neck tumorsfail radiation therapy, it is conceivable that there is acause-and-effect relationship between the lack of functional wtp53 foundin a large number of SCCHN and this observed RR. The replacement ofwtp53 may, therefore, result in the sensitization of these tumors toconventional radiotherapy.

2.5×10⁶ JSQ-3 cells were injected subcutaneously on the lower back abovethe tail of 4-6 week old female athymic nude mice (NCr nu/nu). When thetumors reached the appropriate size, i.v. injection of LipF(A)-p53,pCMVp53 or LipF(A)-pRo, at 8 μg DNA/400 μl 5% dextrose/mouse, were giventwice weekly for a total of 5 injections. 48 hours after the initiali.v. injection, the animals were secured in a lead holder whichpermitted only the tumor area to be irradiated, and the firstfractionated dose of 2.5 Gy of ¹³⁷Cs ionizing radiation administered.Thereafter, the animals were given 2.5 Gy every 48 hours to a total doseof 25 Gy. For comparison, a group of untransfected, as well as a groupof mice receiving LipF(A)-p53, received no radiation.

Athymic nude mice bearing subcutaneous JSQ-3 tumors of approximately25-40 mm³ were intravenously injected, via the tail vein, withLipF(A)-p53 twice weekly (a total of 5 injections) and the tumor areaonly exposed to fractionated doses of gamma radiation (a total of 25Gy). To determine if the transfected p53 protein was expressed in thetumors, one tumor from the untransfected, LipF(A)-p53 and LipF(A)-pRogroups was resected during the course of the experiment (after 3injections and 12.5 Gy). A high level of exogenous p53 protein wasmanifest in the LipF(A)-p53 treated tumor, confirming that thefolate-cationic liposome complex was able to deliver systemically thewtp53 gene to the tumors. Treatment with radiation alone had only alimited effect on the tumors in the untransfected animals. I.V.injection of pCMVp53 plasmid DNA, or LipF(A)-pRo, in combination withionizing radiation, initially induced some inhibition of tumor growth.However, analogous to clinical circumstances, these tumors, like thosein the untransfected animals, begin to re-grow after cessation of theradiation treatment. Although treatment with LipF(A)-p53 alone was ableto inhibit tumor growth for a period of time during and even after theend of the i.v. infections, these tumors also began to increase in sizewithin two weeks of the last i.v. injection. In contrast, 75% of thetumors which received the combination of LipF(A)-p53 plus radiationregressed completely, showing no signs of reoccurrence even 130 dayspost-radiation treatment. Moreover, the other 25% displayed only minimalresidual tumor, which was static at less than 10% of the original tumorvolume over the course of the experiment. Histologic examination of thisresidual mass showed that it represents mature scar with noproliferative tumor cells present.

Currently, after more than one year after cessation of treatment, thecontrol animals have all either died or been humanely euthanized due totumor burden. However, there is still no sign of tumor regrowth inanimals that received the combination treatment.

Similar results were obtained from another, independent experiment, inwhich the initial tumor volumes were between 25 and 60 mm³. Here again,approximately one year post-irradiation, no tumor regrowth is evident inthe animals which received the combination treatment.

This is the first demonstration of total tumor regression mediated by asystemically delivered liposome-p53 complex. These in vivo studiesdemonstrated that the combination of systemic LipF(A)-p53 gene therapyand conventional radiotherapy was markedly more effective than eithertreatment alone.

EXAMPLE 11 Sensitization of JSQ-3 Xenograft Tumors to Radiation by theSystemic Delivery of LipT(A)-p53

In this example we demonstrate the use of the systemically deliveredtransferrin-liposome-therapeutic molecule as a method of cancertreatment. In this particular example, the therapeutic molecule is thenormal human wild-type p53 gene (pCMVp53).

2.5×10⁶ JSQ-3 cells were injected subcutaneously on the lower back abovethe tail of 4-6 week old female athymic nude mice (NCr nu/nu). 7-10 dayslater, the tumors grew to approximately 40-50 mm³ at the injection site.Freshly prepared LipT(A)-p53 or LipT(A)-pRo containing 8 μg DNA in 300ml 5% dextrose were intravenously injected per mouse, via tail veintwice per week, for a total of 5 injections. 48 hours after the initiali.v. injection, the animals were secured in a lead restraint so thatonly the tumor area was exposed to gamma-irradiation, and the firstfractionated dose of 2.5 Gy of ¹³⁷ Cs ionizing radiation administered.Thereafter, the animals were given 2.5 Gy every 48 hours to a total doseof 25 Gy. For comparison, a group of untransfected, as well as a groupof mice with LipT(A)-p53 injection receiving no radiation were used ascontrols. The tumor sizes were measured weekly in a blinded manner.

Two independent experiments with SCCHN (JSQ-3) xenograft tumors werebeen performed with similar results. In the first, mice bearingsubcutaneous JSQ-3 tumors of approximately 25-40 mm³ were injected, viathe tail vein, with LipT(A)-p53 twice weekly (a total of 5 injections)and only the tumor area exposed to fractionated doses of gamma radiation(a total of 25 Gy). Short-term radiation effects on tumor growth wereevident in cells transfected using the control LipT(A)-CMVpRo. There wasonly minimal tumor growth inhibition in the animals that received theLipT(A)-CMVp53 without radiation. In contrast, all of the tumors thatreceived the combination of LipT(A)-CMVp53 plus radiation exhibitedvirtually complete regression, showing no signs of reoccurrence even 153days post-radiation treatment. By this time, the tumor-bearing animalsin control groups had died or were humanely euthanized due to excessivetumor burden. However, at one year post-irradiation the combinationtreatment group (p53 and radiation) still showed no sign of tumorregrowth. As in the case of animals treated with the combination ofLipF(A)-p53 and radiation, by one month post-treatment, only scar tissueand a few invading Langerhan's cells were present in the residual tissueat the site of the original tumor in an animal that received thecombination treatment. Similar results were observed in a second in vivoexperiment.

EXAMPLE 12 Effect of the Combination Therapy in a Second Cancer Model

This example illustrates that the efficacy of this novel combination ofliposome-mediated, tumor-targeted p53 gene therapy and conventionalradiotherapy is not limited to SCCHN, thereby increasing the clinicalrelevance of this system. The effect of the combination offolate-targeted, liposome-mediated p53 gene therapy and radiation onhuman prostate cell line DU145 in vivo was evaluated. Thisadenocarcinoma cell line was originally derived from a lesion in thebrain of a patient with widespread metastatic carcinoma of the prostateand is reported to carry mtp53. We have found this cell line to beresistant to gamma-radiation killing (D₁₀=5.8±0.22 Gy), one of theprimary forms of adjuvant therapy for this disease. Earlier in vitroexperiments suggested that replacing the neutral lipid DOPE withcholesterol may result in increased transfection efficiency in thisdistinct tumor cell type. Based upon the luciferase activity, we foundthat LipF(D) gave more than a four-fold increase in transfectionefficiency as compared to LipF(A) in DU145 cells. Therefore, micebearing DU145 tumors of approximately 70 mm³ were i.v. injected via thetail vein with LipF(D)-p53 approximately every 5 days (a total of 5injections) and the tumors exposed to fractionated doses ofgamma-irradiation (a total of 25 Gy). In this experiment, a non-folatetargeted liposome-p53 composition (Lip(D)-p53) was also used as acontrol. The results with these prostate tumors were quite similar tothose of the SCCHN tumors. Radiation alone, LipF(D)-pRo plus radiation,and non-targeted Lip(D)-p53 plus radiation had some inhibitory effect ontumor growth during the course of the treatments. However, these tumorsall rapidly increased in size once treatment ceased. In contrast, thecombination of LipF(D)-p53 plus radiation again resulted in long-termregression of the tumors even at day 84 which is 64 days post-treatment.An observed drop in tumor volume of the control group at Day 63 was dueto loss of animals in this group due to tumor burden. A secondexperiment with tumors of approximately 100 mm³ showed analogous resultswith no regrowth observed even 47 days after all treatment had ceased.

EXAMPLE 13 chemosensitization of JSQ-3 to Cisplatin (CDDP) In Vitro

In addition to radiation, chemotherapy is becoming more commonly used inthe treatment of SCCHN. As lack of functional wtp53 has been associatedwith failure to respond to chemotherapy, in this example we examined theeffect of ligand-facilitated liposome mediated wtp53 gene therapy onsensitization of SCCHN cell line JSQ-3 to chemotherapeutic agents. 1×10⁴cells were plated/well of a 96-well plate. 24 hours later, the cellswere transfected with LipT(A)-p53. Two days after transfection,anti-neoplastic agents were added at increasing concentrations (intriplicate). 4-6 days later the XTT cell proliferation assay wasperformed and IC₅₀ values, the drug concentration yielding 50% growthinhibition, calculated. Treatment with as little as 0.2 μg of wtp53 DNAcomplexed to LipT(A) was shown to substantially sensitize JSQ-3 cells toboth CDDP and 5-FU, two drugs frequency employed in adjuvantchemotherapy. While transfection with the LipT(A) complex alone, orLipT(A) carrying wtp53 in the reverse orientation (LipT(A)-pRo) yieldedsome sensitization to CDDP, a 24 fold level of sensitization over thatof the untransfected cells was evident in the LipT(A)-p53 transducedcells. Furthermore, a 15.4 fold sensitization of JSQ-3 to thechemotherapeutic agent 5-FU was also observed after transfection withthe LipT(A)-p53 complex.

EXAMPLE 14 P53-mediated Chemosensitization as Indicated by EnhancedInduction of Apoptosis

This example examines the effect of ligand-liposome mediated wtp53restoration on chemotherapeutic agent induced apoptosis. JSQ-3 cellswere seeded in 6-well plates and transfected with LipT(A)-p53,LipT(A)-pVec (the vector without the p53 gene) or LipT(A) alone, at 1 or2 ug DNA per 2×10⁵ cells. 24hr later, chemotherapeutic agents were addedto each set of plates, at concentrations near the IC₅₀ values for eachcell line. After one additional day incubation, both the attached andfloating cells were collected and stained with AnnexinV-FITC, whichbinds specifically to phosphatidylserine present on apoptotic cells,using an Annexin V-FITC Kit (Trevigen, Inc., Gaithersgurg, Md.)according to the manufacturer's protocol. The stained cells wereanalyzed on FACStar flow cytometer (Becton and Dickinson).

A p53 DNA dose dependent induction of apoptosis was observed in thecells treated with the LipT-mediated wtp53 complex of the invention.Moreover, the addition of chemotherapeutic agents (CDDP, Taxotere, 5-FU)at doses near their IC₅₀ values induced a substantial increase in thepercent of apoptotic cells in the population only in LipT(A)-p53transfected cells, not in the untransfected (UT) and LipT(A) only orLipT(A)-pVec transfected cells. The increase was p53 DNA dose-dependentand correlated with the wtp53 expression levels observed on Westernblots, demonstrating that the chemotherapeutic agents inducedenhancement of apoptosis was proportional to the wtp53 level in cells,i.e., the more wtp53 expressed, the more apoptoses was induced. Theincrease in apoptosis observed after the combination of the LipT(A)-p53plus drug was substantially more than the sum of the chemotherapeuticagent alone (UT plus drug) plus p53 transfection alone (p53 no drug),indicating a synergistic effect when p53 gene therapy was combined withchemotherapeutic agents.

EXAMPLE 15 Chemosensitization of MDA-MB-435 to Cisplatin or DoxorubicinIn Vitro by Ligand-liposome-p53

In the treatment of breast cancer, the failure of a substantial portionof tumors, and their metastases, to respond to adjuvant chemotherapy isa major concern. In this example we examined the ability of the deliverysystem of the invention to sensitize breast cancer cells to currentlyused chemotherapeutic agents.

Human breast cancer cell line MDA-MB-435 was employed. Theligand-liposome complex used was the composition, which had beenoptimized for head and neck squamous cells—a different histological celltype from that found in breast cancer. Transfection with LipT(A)-p53increased the effect of doxorubicin on MDA-MB-435 cells by four fold andthe effect of CDDP by almost 12 fold, as compared to the untransfectedcells. As seen with the SCCHN cells, there was also some sensitizationby the LipT(A)-pRo complex. Here again, although not yet optimized formammary carcinomas, chemosensitization of breast cancer cells bytransfection with LipT(A)-p53 was demonstrated.

Even more striking results were obtained using a composition, LipF(C),tailored for adenocarcinoma, the histological cell type of most breastcancers. As above, the IC₅₀ values were determined by the XTT assay.Transfection with LipF(C)-p53 increased the effect of doxorubicin onMDA-MB-435 cells by 73.6-fold and the effect of Taxol by 31.6-fold, ascompared to the untransfected cells. As seen with the SCCHN cells, therewas also some sensitization by the LipF(C)-pRo complex. These resultsdemonstrate chemosensitization of breast cancer cells by transfectionwith transferrin- and folate-targeted liposome-p53 complex.

EXAMPLE 16 Chemosensitization of Breast Cancer Cells by LipF-p53 GeneTherapy In Vivo

This example shows the ability of the systemically deliveredligand-liposome-therapeutic molecule complex of the invention to be aneffective therapeutic agent against cancer cells in vivo. Mice bearingsubcutaneous mammary fat pad MDA-MB-435 tumors of approximately 100 mm³were i.v. injected via the tail vein with LipF(C)-p53 every 3-4 days fora total of eight injections. Doxorubicin (Dxr) (10 mg/kg) was injectedi.v. weekly for 4 weeks. The combination of LipF(C)-p53 and Dxrsubstantially inhibited the growth of the tumors. In a secondexperiment, two separate liposome compositions [LipF(E) and LipF(C)]were employed. Both demonstrated an effect in combination with Dxr, withthe LipF(C)-p53 composition being superior to that of LipF(E)-p53.

EXAMPLE 17 Optimization of Ligand-liposome Transfection in DifferentCancer Cell Lines

In this example we further explored the ligand-cationic liposome system,preparing a panel of ligand-targeted cationic liposomes to optimize thetransfection efficiency to a variety of human and rodent cancer cells.

Cationic liposomes were prepared as follows:

LipA DOTAP/DOPE 1:1 molar ratio LipB DDAB/DOPE 1:1 molar ratio LipCDDAB/DOPE 1:2 molar ratio LipD DOTAP/Chol 1:1 molar ratio LipE DDAB/Chol1:1 molar ratio LipG DOTAP/DOPE/Chol 2:1:1 molar ratio LipHDDAB/DOPE/Chol 2:1:1 molar ratio

1. Folate series: Each of the above formulations plus 1%-5% folate-DOPEor folate-DSPE.

2. Transferrin series: Each of the above formulations mixed withholo-transferrin in medium or buffer, then mixed with reporter geneplasmid DNA in medium or buffer to form the complex.

The firefly luciferase gene in plasmid pCMVLuc or E. coliβ-galactosidase gene in plasmid pCMVb was used as a reporter gene.

Preparation of DNA-liposame Complexes:

The various DNA-Liposome-Folate complexes was prepared by mixing, inpolypropylene tubes, equal amounts of serum-free medium and the reportergene plasmid DNA in TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0), andequal amounts of serum-free medium with the folate-liposome (LipA-F,LipB-F, LipC-F, LipD-F, LipE-F, tipG-F, LipH-F) in sterile water (2μmol/ml total lipids). After 10-15 min at room temperature, the twosolutions were mixed together and incubated 15-30 min at roomtemperature with frequent rocking. The DNA to lipid ratios inoptimization ranged from 1:0.1 to 1:50 ug/nmol.

The various DNA-Liposome-Transferrin complexes were prepared by theaddition of Tf (iron-saturated, Sigma, 4-5 mg/ml in water, filtered with0.22 mm filter) to serum-free medium. 5-15 min later, cationic liposome(LipA, LipB, LipC, LipD, LipE, LipG, LipH) was added and mixed. After5-15 min. incubation at room temperature with frequent rocking, an equalamount of medium containing reporter gene plasmid DNA was added andmixed, and incubated 15-30 min at room temperature with frequentrocking. The DNA/Lipid/Tf ratios in optimization were in the range of1/(0.1-50)/(0.1-100) μg/nmol/μg.

Cell lines:

Optimization was performed on the following cell lines:

Human squamous cell carcinoma of head and neck: JSQ-3, HN17B, HN22a,HN-38, SCC-25.

Human breast cancer: MDA-MB-231, MDA-MB-435, MDA-MB-453, MCF-7.

Human prostate cancer: DU145, LNCaP, Ln-30, P4-20.

Human ovary cancer: SKOV-3, PA-1

Human pancreatic cancer: PANC-1

Human colon cancer: SW480, LS174T, SK-CO-1

Human glioblastoma: U-87

Human cervical cancer: HTB-34, ME180

Human lung cancer: CALU-3

Human gastric cancer: Hs 746T

Human liposarcoma: SW 872

Human melanoma: SK-MEL-31

Human choriocarcinoma: JEG-3

Human rhabdomyosarcoma: Hs 729T

Human retinoblastoma: Y79

Human normal breast epithelial: Hs578Bst

Human endothelial: HUV-EC-C

Mouse melanoma: B16/F10

Rat prostate cancer: PA-III, AT.61

Rat brain cancer: RT-2

Optimization by Luciferase Assay:

5×10⁴ cells/well were plated in a 24-well plate. 24 hours later, thecells were washed once with medium without serum, 0.3 ml medium withoutserum and antibiotics was added to each well. The freshly preparedLipT-pCMVLuc or LipF-pCMVLuc complexes containing different amounts ofplasmid DNA (up to 1.0 μg in 0.2 ml) medium was added to the cells.After a 5-hour incubation at 37° C. and 5% CO₂, 0.5 ml mediumsupplemented with 20% fetal bovine serum was added to each well. 24hours later, the cells were washed once with PBS, lysed with 100 μl/well1X Reporter Lysis Buffer (Promega), and the expressed luciferaseactivities were measured with Luciferase Assay System (Promega) on aLuminometer. The protein concentration of the cell lysate was measuredusing the Bio-Rad DC Protein Assay Kit (Bio-Rad Laboratories). Theresults were expressed as relative light unit (RLU) per ug of totalprotein.

Optimization by β-Galactosldase Calorimetric Assay:

1×10⁴ cells were plated in each well of a 96-well plate or 5×10⁴cells/well in 24-well plate. 24 hours later, the cells were washed oncewith medium without serum or antibiotics and 100 μl transfectionsolution containing various Amounts of LipT-pCMVb, LipF-pCMVb, or pCMVbalone, were added to each well. After 5 hours at 37° C., an equal amountof medium containing 20% fetal bovine serum was added to each well. 48hours later, the cells were washed once with PBS, and lysed in 1Xreporter lysis buffer (Promega). The cell lysates were treated with 100μl 150 μM 0-nitrophenyl-β-galactopyranoside in 20 mM Tris (pH 7.5)containing 1mM MgCl₂ and 450 μM β-mercaptoethanol at 37° C. for 0.5hour. The reaction was stopped by the addition of 150 μl/well of 1 MNa₂CO₃. The absorbency was determined at 405 nm. Purifiedβ-galactosidase (Boehringer) was used as standard. The results wereexpressed as milli-unit of β-galactosidase equivalent per ug of totalprotein.

Histochemical Staining:

For histochemical studies of ligand-liposome-pCMVb transfection, cellsat 60% confluence (in a 24-well plate) were transfected for 5 hours asdescribed above. After an additional 2 days in culture, the cells werefixed and stained with X-gal. Transfection efficiency was calculated aspercentage of blue-stained cells.

Transfection Efficiencies of the Different Liposome Compositions withDifferent Cell Lines:

As shown in Table 2, LipT(A) and LipT(D) demonstrated the highesttransfection efficiency for JSQ-3 cells, 3-8 fold more efficient thanother liposome formulations. LipT(D) was the most efficient for bothMDA-MB-435 and DU145. At the ratio of 1/12/15 (DNA μg/Lip nmol/Tf μg) orhigher, LipT(D) gave high efficiency to JSQ-3 and LipT(A) to MDA-MB-435cells, but cytotoxicity became obvious. More importantly, when preparingTf-Lip-DNA complexes for in vivo experiments, the complex at this ratioor higher (lipids) tends to precipitate, the solution of the complextends to become cloudy (i.e., not as clear as solutions prepared atlower ratios) and not stable. Therefore, the preferred ratio of LipT is1/10/12.5 (DNA μg/Lip nmol/Tf μg).

TABLE 2 Liposomes Ratio** DU145 MDA-MB-435 JSQ-3 LipT(A) 1/6/7.5 0.621.18 24.62 1/8/10 1.54 2.90 76.07 1/10//12.5 3.05 2.32 117.64 1/12/151.50 14.56 81.09 LipT(B) 1/6/7.5 1.06 6.35 44.11 1/8/10 0.97 5.91 36.451/10/12.5 0.78 N/A 43.00 1/12/15 0.28 5.90 38.98 LipT(C) 1/6/7.5 0.0430.66 2.80 1/8/10 0.087 1.63 7.35 1/10/12.5 0.33 2.59 16.59 11/12/15 0.253.48 17.29 LipT(D) 1/6/75 0.076 4.00 1.88 1/8/10 0.26 7.43 3.431/10/12.5 0.92 9.63 42.20 1/12/15 3.06 13.44 124.60 LipT(E) 1/6/7.5 0.547.56 9.46 1/8/10 0.87 5.31 8.96 1/10/12.5 1.12 4.52 20.91 1/12/15 1.336.21 27.95 Plasmid Alone 0.0001 0.0034 0.0001 *×10⁶ RLU/mg protein**Ratios of DNA μg/Lip nmol/Tf μg

Similar to transferrin, LipF(A) and LipF(C) provided the best resultsfor JSQ-3 cells, 2 to 8-fold more efficient than other liposomeformulations (Table 3). Interestingly, folate-liposomes give totallydifferent patterns of efficiencies compared with Tf-liposomes in bothMDA-MB-435 and DU145 cells, and in other cell lines as well. LipF(C)provided the best results for MDA-MB-435 and LipF(E) provided the bestresults for DU145 (Table 3). Similar results with less efficiency wereobtained in some cancer cell lines transfected with

TABLE 3 MDA-MB- JSQ-3 DU145 DUI145 435 0.5 ug Liposomes 0.25 ug DNA 0.5ug DNA 0.5 ug DNA DNA LipF(A) 1/6** 0.05 1/8 0.31 0.58 2.16 76.90 1/100.16 0.29 0.59 77.96 1/12 0.18 LipF(B) 1/6 0.42 1/8 1.27 2.68 2.26 44.801/10 1.03 1.94 1.71 42.15 1/12 1.61 LipF(C) 1/6 0.10 1/8 0.44 1.14 3.5836.27 1/10 0.54 1.15 1.62 83.88 1/12 0.35 LipF(D) 1/6 0.05 1/8 0.05 0.531.07 25.95 1/10 0.38 0.74 0.64 34.47 1/12 0.20 LipF(E) 1/6 2.71 1/8 2.082.23 0.98 12.12 1/10 1.63 2.95 1.07 23.91 1/12 1.60 Plasmid 0.27 × 10⁻⁶0.13 × 10⁻³ 0 0 *×10⁶ RLU/mg protein **Ratios: DNA μg/Lip nmol

Table 4 shows the preferred ligand-liposome formulations for some of thecell lines we have tested in vitro using the ligand-liposome systemdisclosed in the invention. It should be noted that the optimalcompositions for in vitro transfection are not is necessarily theoptimal ones for in vivo transfection. But it tends to be that the invitro preferred compositions are a good starting point leading to thepreferred compositions for in vivo. Therefore, in vivo optimizationusing nude mouse xenograft models is necessary before the in vivosystemic gene therapy experiments, as disclosed in the invention.

TABLE 4 Cell Line Tf-liposome Folate-liposome JSQ-3 LipT(A),(D)LipF(A),(C) HN 17B LipT(B) HN 22a LipT(A) HN 38 LipT(B) SCC-25 LipT(A)SCC-25cp LipT(A) MDA-MB-231 LipT(E) MDA-MB-435 LipT(D),(A) LipF(C),(B)MDA-MB-453 LipT(C) DU 145 LipT(D),(H) LipF(E) P4-20 LipT(A) SKOV-3LipT(D),(B) PA-1 LipT(A) PANC-1 LipT(D),(H) LipF(D),(A) SW 480 LipT(A)LS 174T LipF(D) SK-CO-1 LipF(E) U-87 LipT(D),(A) HTB-34 LipT(C),(A)LipF(C) ME 180 LipF(E) CALU-3 LipF(D) HS 746T LipF(E) HS 578 Bst LipF(E)HUV-EC-C LipF(E) B16 F10 LipT(A),(C) LipF(E) JEG-3 LipF(B) HS 729TLipF(B) Y79 LipF(D) PA-III LipF(C),(H) AT6.1 LipT(H) LipF(H) RT-2LipF(B)

Effect of Serum on the Transfection Efficiency of Ligand-liposomes:

LipT(D) had the highest level of transfection efficiency with humanglioblastoma cell line U-87 without serum. However, in the presence of10% serum its transfection efficiency was substantially reduced, whileLipT(A) was most efficient in the presence of serum for this cell line.For human pancreatic cancer cell line PANC-1, serum appeared to enhancethe transfection with some liposome compositions, with LipT(H)displaying the highest level of efficiency. Here again, we observeddifferent transfection efficiency patterns in different cell lines, anddifferent effects of serum on transfection efficiency. For the purposeof in vivo transfection, serum effects should be considered duringoptimization.

EXAMPLE 18 Chemosensitization of Other Cell Lines by Tf- orFolate-Liposome-Mediated Wtp53 Gene Therapy In Vitro

This example summarizes part of the in vitro p53-mediatedchemosensitization experiments (XTT assays) performed on the cell linesfor which transfection with transferrin-coupled or folate-coupledliposomes is described in Example 17. The data presented in thefollowing table demonstrate that both LipT- and LipF-mediated p53 genetransfection can sensitize these tumor cells to chemotherapeutic agents.The chemosensitization effect is dependent upon the liposome used andthe p53 DNA dose. VIN=vinblastine; DXR=doxorubicin; CDDP=cisplatin. FoldSensitization is calculated from the individual IC₅₀ values. DNA dose=ugof DNA applied per well (approximately 1×10⁴ cells/well in a 96-wellplate).

DNA IC50 Fold Sensitization Cell line Drug Liposome Dose p53 Vector Liponly UT p53 vs Vector p53 vs UT MDA-MB-231 Vln LipF(B) 0.15 0.42 1.071.23 1.57 2.6 3.8 Vln LipF(A) 0.15 0.50 1.42 1.47 1.81 2.9 3.6 VlnLipF(C) 0.15 0.48 1.69 1.47 1.81 3.5 3.8 DXR LipF(B) 0.15 0.07 0.24 0.270.27 3.6 4.0 DXR LipF(A) 0.15 0.11 0.24 0.22 0.20 2.2 1.8 DXR LipF(C)0.15 0.02 0.09 0.30 0.27 4.5 13.5 Taxol LipF(C) 0.12 2.90 11.70 11.7054.10 4.0 18.7 Taxol LipF(C) 0.08 6.10 10.00 10.00 52.00 1.6 8.5 TaxolLipT(C) 0.12 14.70 46.70 10.00 52.00 3.2 3.5 MDA-MB-435 DXR LipF(C) 0.120.01 0.74 0.74 0.89 62.9 76.1 DXR LipF(C) 0.08 0.01 0.58 0.76 0.93 46.373.5 DXR LipT(B) 0.10 0.01 0.54 0.79 0.85 54.0 85.0 Taxol LipF(C) 0.121.12 10.79 34.15 39.80 9.6 35.5 Taxol LipF(C) 0.08 1.36 14.13 36.8742.99 10.4 31.6 Taxotere LipF(B) 0.10 0.11 3.75 13.50 10.00 34.7 92.6Taxotere LipT(B) 0.08 0.07 0.80 3.10 3.60 10.8 48.7 JSQ-3 TaxotereLipF(A) 0.10 0.29 1.73 2.13 2.53 6.0 8.7 Taxotere LipF(C) 0.10 1.36 3.983.40 13.59 2.9 10.0 Taxotere LipF(C) 0.08 0.79 3.98 3.40 13.59 5.0 17.2Taxotere LipF(B) 0.10 0.86 1.85 13.59 14.68 2.2 17.1 Taxol LipF(C) 0.101.70 3.40 6.30 15.85 2.0 9.3 Taxol LipF(C) 0.08 1.47 3.40 6.30 15.85 2.310.8 Taxol LipF(B) 0.10 0.86 3.16 17.11 17.11 3.7 19.9 DU145 TaxotereLiPF(C) 0.10 0.86 6.60 1.35 41.97 7.7 48.8 Taxotere LipF(C) 0.08 0.3241.97 130.3 Taxotere LipF(B) 0.10 0.56 3.40 14.68 39.80 6.1 71.1 TaxolLipF(C) 0.10 0.71 4.20 4.80 13.20 5.9 18.6 Taxol LipF(B) 0.10 1.40 5.5013.50 16.30 3.9 11.6 PANC-1 CDDP LipF(B) 0.10 5.05 13.33 14.86 18.43 2.63.6 Taxotere LipF(A) 0.15 1.63 10.36 11.50 6.4 7.1 Taxotere LipF(B) 0.150.14 1.30 10.00 12.30 9.3 87.9 Taxotere LipF(C) 0.20 0.15 1.70 1.9012.30 11.3 82.0 Gemzar LipT(B) 0.20 0.05 0.81 0.80 16.2 15.1 U87 GemzarLipF(B) 0.10 0.28 1.07 0.76 1.28 3.9 4.7 Gemzar LipF(C) 0.10 0.50 1.151.20 1.23 2.3 2.5 Gemzar LipT(A) 0.20 0.20 1.00 1.00 1.00 5.0 5.0

EXAMPLE 19 Effect of the Combination of Systemically Delivered LipF-p53and Chemotherapy on the Growth of DU145 Xenografts In Vivo

Chemotherapy is becoming more commonly used in the treatment of prostatecancer. Lack of functional wtp53 has been associated with failure torespond to chemotherapy. This example examines the effect of thecombination of ligand-liposome-p53 and chemotherapeutics on the growthof prostate tumor xenografts in vivo.

Mice bearing subcutaneous DU145 xenograft tumors of approximately 100mm³ were injected, via the tail vein, with a ligand-liposome p53 complexusing folate as the targeting ligand (LipF(B)-p53). This liposomecomplex was administered twice/week (to a total of 5 injections) alongwith the chemotherapeutic agent docetaxel at a dose of 10 mg/kg.Treatment of the animals with neither the LipF(B)-p53 complex alone, nordocetaxel alone had any substantial effect on tumor growth. However,treatment with the combination of the systemically delivered LipF(B)-p53of the invention plus docetaxel led to substantial tumor regression.Though the complex used was not been completely optimized for prostatetumor cells, these findings strongly support the ability of systemicallydelivered, targeted-liposomes to deliver wtp53 to the tumors resultingin their sensitization to conventional therapeutics.

EXAMPLE 20 Effect of the Combination of Systemically Delivered LipF-p53and Chemotherapy on the Growth of PANC-1 Xenografts In Vivo

This example demonstrates the effect of the combination ofligand-liposome-p53 and chemotherapeutics on the growth of pancreaticcancer xenografts in vivo. Xenograft tumors of pancreatic cancer cellline PANC-1 were induced by the subcutaneous inoculation of greater than1×10⁷ cells onto athymic nude mice. When the tumors reachedapproximately 500-1000 mm³, the tumors were excised and minced intosmall (<1 mm) pieces. These freshly prepared tumor pieces (suspended inPBS) were inoculated subcutaneously (using a 14 g needle) onto theflanks of athymic nude mice. When the tumors reached an average of 100mm³ in volume, treatment was begun. The animals received, viaintravenous injection, LipF(B)-p53. This liposome complex wasadministered twice/week to a total of 7 injections. The chemotherapeuticagent gemcitabine was also administered intraperitoneally at a dose ofeither 60mg/kg or 120mg/kg twice weekly. A total of 13 gemcitabineinjections were administered. One group of animals also received twiceweekly intratumoral injections of LipF(B)-p53 (a total 6) in addition tothe intravenous administration of the LipF(B)-p53 and gemcitabine. Thecontrol groups of animals that received no treatment, only gemcitabine,only LipF(B)-p53, or LipF(B) complexed to the pCMV vector without p53(LipF(B)-Vec) were euthanized due to tumor burden by day 54. Incontrast, the three groups of animals receiving the combination ofLipF(B)-p53 and gemcitabine showed substantial growth inhibition oftheir tumors, even 12 days after the end of treatment. This wasparticularly evident in the group that received both i.v. and i.tinjections. Therefore, once again, using another tumor model, thecombination of systemically delivered ligand-liposome-therapeuticmolecule and chemotherapeutic agent was found to be substantially moreeffective than currently available therapies.

EXAMPLE 21 Chemosensitization of Tumor Cells by Ligand-Targeted,Liposome-Mediated Antisense Oligonucleotides In Vitro and In Vivo

This example demonstrates the ability of the systemically administeredligand-liposome-therapeutic molecule delivery system of the invention todeliver small oligonucleotides as the therapeutic molecule. Further,this example demonstrates the ability of the systemically administered,ligand-liposome-delivery of the small oligonucleotides to sensitize thecontacted tumor cells to chemotherapeutic agents.

Optimization of the Folate-liposome (LipF) Composition for Various TumorCell Types:

Starting with the ligand-liposome complex derived for SCCHN cell linesand described above, further ligand-liposome compositions optimized fordelivering anti-sense HER-2 (AS-HER-2) oligonucleotides to tumor cellswere developed. The AS-HER-2 oligonucleotide was a 15-mer complementaryto a sequence near the initiation codon of the HER-2 gene. (Pirollo etal., BBRC 230, 196-201 (1997)).

Saturation of Liposomes by Oligonucleotides:

Multiple new folate-liposomes (LipF) compositions were produced byvarying the cationic and neutral lipid in the complex. Helper lipidswere also included in some compositions. The ratio of cationic toneutral lipid was also varied. Using ³²P-labled AS-HER-2oligonucleotide, we determined the ratio of liposome to oligonucleotidethat gave optimal binding of the oligonucleotide to the variouscompositions. An example of these studies is shown in the followingtable where a comparison is made between LipF compositions B and Cversus Liposome A, which is the LipF composition optimized for SCCHN.

Ratio Lip:Oligo Liposome A Liposome B Liposome C  1:10 23% 51.6% 44.25%1:1 87.7%   77.9% 61.97% 5:1 90% 90.7%  72.6% 10:1  93%   98%  86.7%25:1  100%   100%   100%

There is clearly a difference in the oligonucleotide binding between thethree compositions. Nevertheless, complete saturation is achieved withall three at a liposome:oligonucleotide ratio of 25:1. However, asubstantial amount of toxicity was evident at this ratio. It is alsoevident from these data that for different liposome compositions, theoptimal ratio is dramatically different.

AS-HER-2 Oligonucleotide Uptake by Tumor Cell Lines with Various LipFCompositions:

Transfection experiments were performed with the LipF compositions andhuman breast cancer cell line MDA-MB-435, SCCHN cell line JSQ-3,prostate tumor cell line DU145 and pancreatic tumor cell line PANC 1, todetermine the transfection efficiency of each LipF composition. Thoseused were the four compositions (designated B-E) which were found tohave the most efficient binding of the oligonucleotide. The two molarratios of Liposome:Oligonucleotide used initially in these studies were10:1 and 25:1, those found (see above) to possess the highestoligonucleotide binding levels. However, a ratio of 25:1 was found to betoxic to the cells. Therefore, the remainder of the experiments wereperformed using a ratio of 10:1 (liposome:oligonucleotide).Transfections, using ³²P labeled AS-HER-2, were performed as previouslydescribed for LipF(A)-p53 for SCCHN. However, after twenty hoursincubation at 37° C., the media was removed and the cells washed fivetimes with PBS. The media and washes were combined and the amount ofunincorporated label ascertained. The amount of cell associated³²P-labelled anti-HER-2 oligonucleotide was determined by comparing the³²P level within the cells versus the unincorporated oligonucleotide. Inthese studies LipF(A) is the composition originally optimized for SCCHN.As shown in the following table, LipF composition B yielded the highestlevel of transfection efficiency in MDA-MB-435 breast cancer cells,while LipF composition E was better for both DU145 and PANC I.Therefore, LipF composition B [LipF(B)] was used for the remainder ofthe studies with MDA-MB-435, described below.

Liposome Liposome Liposome Liposome Liposome CELL LINE A B C D E MDA-MB-112 280 108 242 137 435 JSQ-3 184 100 8 125 205 DU145 93 158 130 403 705PANC1 330 490 407 398 731

Oligonucleotide Concentration was 2 μM

Molar Ratio of Liposomes:Oligonucleotide was 10:1

Stability of LipF(B)-AS-HER-2 In Vitro and in Blood:

As a goal of these studies was to develop a systemic delivery system forantisense oligonucleotides, it was important to determine the stabilityof the LipF(B)-AS-HER-2 complex in serum. Therefore, the complex wasadded to 50% serum and incubated at 37° C. At various times from 0-24hours, samples were taken, the oligonucleotides labeled with ³²P andpercent degradation assessed by PAGE. No degradation of the AS-HER-2oligonucleotide was found when complexed to LipF(B) for 24 hours. Incontrast, over 50% of the free oligonucleotide was degraded as early as6 hours, with virtually complete degradation by 24 hours.

The stability was also examined in mouse blood, a setting more analogousto the in vivo situation. Even after 24 hours, more than 75% of thecomplexed oligonucleotide remained intact. Therefore, it was concludedthat the folate targeted delivery system should protect theoligonucleotide long enough in circulation to allow it to effectivelyreach the tumor cells.

In Vitro Chemosensitization of Cancer Cells by LipF-AS-HER-2:

The ability of the LipF(B)-delivered AS-HER-2 to sensitize MDA-MB-435,JSQ-3, DU145 and U87 (Human glioblastoma) cells to chemotherapeuticagents was evaluated. Sensitivity was determined using the XTT cellproliferation assay. Transfection with LipF(B)-AS-HER-2 substantiallyincreased the killing effect of docetaxel upon the 435 cells. Comparisonof the cells treated with LipF(B) mediated AS-HER-2, to that of cellstreated with a LipF(B) control oligonucleotide (SC) indicated a greaterthan 30 fold increase in sensitization of 435 cells to taxotere. Incontrast, only a 2.5 fold level of sensitization was evident aftertransfection with AS-HER-2 using the commercial Lipofectin (LifeTechnologies, Inc.). Treatment of JSQ-3 cells with LipF(E)-AS-HER-2increased the effect of docetaxel almost 25 fold. Moreover, the effectof cisplatin (CDDP) on JSQ-3 cells was also increased by greater than 17fold after treatment with AS-HER-2 complexed to transferrin-targetedLiposome A (LipT(A)). A two fold increase in sensitization of DU145cells to docetaxel was seen after treatment with LipF(E)-AS-HER-2. Humanglioblastoma cell line U87 showed a greater than 8 fold increase inchemosensitivity to the drug gemcitabine after treatment withLipF(B)-AS-HER-2.

To further demonstrate the use of the targeted liposome complex as avector for antisense gene therapy delivery, the ability of LipF(B)carrying an anti-RAS oligonucleotide (AS-RAS, an 11 mer sequencecomplementary to the sequence near the initiation codon of the gene) tosensitize PANC I pancreatic carcinoma cells to docetaxel was examined.Here also a greater than 70 fold increase in drug sensitivity wasinduced by treatment with LipF(B)-AS-RAS. The data showed thatLipF(B)-mediated antisense gene therapy can lead to a substantialincrease in the effectiveness of chemotherapeutic agents in previouslyresistant human cancer cells.

In Vivo Studies

The ability of the LipF(B)-AS-HER-2 to target and sensitize preexistingMDA-MB-435 xenograft tumors to the chemotherapeutic agent docetaxel invivo was examined by assessing tumor regression as well as tumor growthinhibition. Female athymic (Ncr nu/nu) mice carrying MDA-MB-435 mammaryfat pad xenograft tumors of approximately 70 mm³ were intravenouslyinjected, via the tail vein, with LipF(B)-AS-HER-2 (at approximately 0.6mM of oligonucleotides) every other day to a total of 11 injections. Atotal of 11 intravenous doses of docetaxel (approximately 20 mg/kg/doseevery other day) were also administered to the animals. Dramatic growthinhibition of the tumors was evident in the animals receiving thecombination of LipF(B)-AS-HER-2 and docetaxel. In contrast, only minimalgrowth inhibition was evident in those mice receiving just AS-HER-2.Moreover, while there was some docetaxel effect, these tumors began torapidly increase in size after the cessation of treatment. Therefore,the systemically delivered, targeted liposome delivery of antisenseoligonucleotides, in this case AS-HER-2, was clearly able to sensitizethese tumors to the chemotherapeutic agent, strongly inhibiting tumorgrowth almost three weeks after the end of treatment.

EXAMPLE 22 Targeting of Adenovirus by Transferrin-Liposomes

Improving the efficiency and specificity of gene transfer remains animportant goal in developing new strategies for gene therapy.Adenoviruses (Ad) are highly efficient vectors, but they are limited bylack of tumor targeting specificity and substantial immunogenicity. Ithas been reported that cationic lipids can form non-covalent complexwith adenovirus and enhance gene transfer efficiency. But cationiclipids themselves still lack target specificity.

In this example we demonstrated that the ligand-liposome vector of theinvention can also form a complex with adenovirus particles, therebyenhancing their gene transfer efficiency, and more substantially, theirtargeting specificity. Moreover, the use of theligand-liposome-therapeutic molecule delivery system of the invention,when the therapeutic molecule is an intact adenovirus particle, allowsefficient tumor cell targeting and systemic administration oftherapeutic adenovirus for gene therapy, another novel approach to genetherapy.

Preparation of Transferrin-Liposome-Adenovirus Complex

Replication-deficient adenovirus serotype 5 containing E. coliβ-galactosidase gene LacZ under CMV promoter, Ad5LacZ, was used in thestudy. The Ad5LacZ, at 1.1×10¹² particles (pt)/ml, or 5.5×10⁹ plaqueforming unit (pfu)/ml, in PBS (pH 7.4) plus 3% sucrose was used in thestudy. Holo-transferrin (Tf, iron-saturated, Sigma) was dissolved inwater at 4-5 mg/ml and filtered with 0.22 mm filter. Tf was firstdiluted to 0.5 mg/ml in 10 mM HEPES buffer (pH 7.4) following whichdifferent amounts of Tf were added to 50 μl HEPES buffer inmicrocentrifuge tubes and mixed well. After 5-10 min incubation at roomtemperature, Lip(A) (DOTAP:DOPE 1:1 molar ratio) at 0.1 nmol/μl wasadded to the tubes so that the lipid/Tf ratios ranged from 1 nmol/1-10g. The solutions were mixed well and incubated at room temperature for5-10 min. 1×10⁶-1×10⁷pt adenovirus was added to each tube so that thecationic lipid/adenovirus ratios ranged from 1×10³ to 1×10⁷ lipidmolecules/pt. The samples were incubated at room temperature 10-15 minand then 150 μl EMEM without serum was added to each.

In Vitro Adenoviral Transduction

5×10⁴ JSQ-3 cells/well were plated in a 24-well plate. 24 hours later,the cells were washed once with EMEM without serum, 0.3 ml EMEM withoutserum or antibiotics was added to each well. The Ad5LacZ or Tf-Ad5LacZcomplexes at different ratios in 200 μl EMEM was added to each well induplicate. The virus to cell ratio ranged from 20 to 2000 viralparticles/cell (pt/cell). After 4 hours incubation at 37° C., 5% CO2,with occasional rocking, 0.5 ml EMEM with 20% serum was added. After 2days culture, the cells were washed once with PBS, lysed in 1X reporterlysis buffer (Promega). The cell lysates were centrifuged, transferredto a 96-well plate in duplicate, incubated with 100 μl of 150 μM0-nitrophenyl-β-galactopyranoside in 20 mM Tris (pH 7.5) containing 1 mMMgCl₂ and 450 μM β-mercaptoethanol at 37° C. for 30 min. The reactionwas stopped by the addition of 150 μl/well of 1 M Na₂CO₃. The absorbencywas determined at 405 nm in an ELISA plate reader. Purifiedβ-galactosidase (Boehringer) was used to produce a standard curve. Theresults were expressed as milliUnit (mU) of β-galactosidase equivalentper mg of total protein.

Histochemical Staining

For histochemical studies of LipT-Ad5LacZ transduction, 60% confluentcells in 24-well plate were transfected for 5 hours with transfectionsolutions as described above. After an additional 2 days in culture, thecells were fixed and stained with X-gal. Transfection efficiency wascalculated as percentage of blue-stained cells.

At a viral dose of 500 pt/cell or 2.5 MOI (multiplicity of infection, orpfu/cell), 10 mU/μg protein of reporter gene product β-galactosidase wasexpressed by Ad5LacZ alone. The transferrin-liposome complexed virus,LipT-Ad5LacZ, at a ratio of 1×10⁴ cationic lipid molecules/pt yielded areporter gene expression of 23.5 mU/μg protein. LipT-Ad5LacZ at 1×10⁵lipid molecules/pt yielded 30.7 mU/μg expression, while LipT-Ad5LacZ at1×10⁶ molecules/pt resulted in 30.8 mU/μg expression. This represents a2.4, 3.07 and 3.08-fold, respectively, increase in gene transductionthan Ad5LacZ alone. Saturation was apparently reached at 1×10⁵ lipidmolecules/pt.

At a dose of 1000 pt/cell (or 5 MOI), LipT-Ad5LacZ at 10⁴ lipids/pt,demonstrated a 2.6-fold increase in reporter gene expression, whileLipT-Ad5LacZ at 10⁵ lipids/pt gave 2.8-fold increase, and LipT-Ad5LacZat 10⁶ lipids/pt, a 3.8-fold higher level of reporter gene expressionthan Ad5LacZ alone. Liposome complex without transferrin gave onlylimited enhancement. Therefore, the optimal ratios of LipT-Ad5LacZcomplex appeared to be about 10-1000 cationic lipids/Tf molecule, andabout 10⁴-10⁷ cationic lipids/pt, preferably about 15-50 cationiclipids/Tf molecule and about 10⁶ cationic lipids/pt. If the lipid/ptratio is too high, precipitation can occur.

Histochemical staining showed that Ad5LacZ alone gave 20-30%transduction efficiency while transferrin-liposome complexed adenovirusLipT-Ad5LacZ at 10⁶ lipids/pt gave 70-90% efficiency.

Other liposome compositions were tested for their ability to complexadenovirus. LipT(B) (DDAB/DOPE, 1:1 molar ratio) and LipT(D)(DOTAP/Chol, 1:1 molar ratio) showed enhanced adenoviral genetransduction into human prostate cancer cell line DU145.

The ligand-liposome delivery system of the invention was also complexedto replication-deficient adenovirus serotype 5 containing 1.7 kb ofhuman wt p53 gene (LipT(D)-Adp53). The LipT(D)-Adp53 complex wasintravenously injected into nude mice bearing DU145 prostate cancerxenograft tumors. Western analysis (performed 72 hours post injection)of the tumor demonstrated the presence of additional bands representingthe exogenous human wt p53 protein present in the tumor tissue. Noadditional, exogenous wt p53 sequences were evident in the normaltissues (e.g. liver, lung or spleen) of the treated animal. These datashow that the ligand-liposome-therapeutic molecule delivery system ofthe invention is capable of delivering adenovirus as the “therapeuticmolecule” specifically to tumor tissue following systemicadministration.

The above results demonstrated that transferrin-cationic liposomes cancomplex adenovirus and substantially enhance adenoviral genetransduction. The administration of ligand-liposome-adenovirus complexesrepresents a novel approach to human gene therapy.

EXAMPLE 23 Transferrin-Liposome-targeted Retroviral Geno Transduction

Retroviral vectors are one of the most widely used gene therapy vectorsin clinical trials. As with adenoviral vectors, retroviral vectors arelimited by poor tumor specificity and significant immunogenicity. Inthis example we demonstrate that, like adenovirus, the ligand-liposomeof the invention can form a complex with retrovirus particles therebyenhancing their gene transfer efficiency, and more significantly, theirtargeting specificity. Moreover, the use of theligand-liposome-therapeutic molecule delivery system of the invention,when the therapeutic molecule is an intact retrovirus particle, allowsefficient tumor cell targeting and the systemic administration ofretroviral vectors for gene therapy.

Replication-deficient retrovirus containing the E. coli LacZ gene,RvLacZ, at 1×10¹⁰ particles (pt)/ml containing 3×10⁷ transforming unit(TU)/ml, was employed in this study. Holo-transferrin (Tf,iron-saturated, Sigma) was dissolved in water at 4-5 mg/ml and filteredwith 0.22mm filter. The LipT-RvLacZ complex was prepared similarly tothat of the LipT-Ad5LacZ described above in Example 21. Briefly, Tf wasfirst diluted to 0.5 mg/ml in 10 mM HEPES buffer (pH 7.4). Differentamounts of Tf were added to 50 μl HEPES buffer microcentrifuge tubes andmixed well. After 5-10 min incubation at room temperature, cationicliposome Lip(A) (DOTAP:DOPE 1:1 molar ratio). The solutions were mixedwell and incubated at room temperature for 5-10 min. 1×10⁶-1×10⁷ptretrovirus were added to each tube so that the cationic lipid/retrovirusratios ranged from 1×10³ to 1×10⁷ lipid molecules/pt. The samples wereincubated at room temperature 10-15 min and 150 μl EMEM without serumwas added to each. In vitro retroviral transduction was performed asdescribed in Example 21. The virus to cell ratio ranged from 100 to 2000viral particles/cell (pt/cell).

At a dose of 1000 pt/cell or 3 MOI (multiplicity of infection, orTU/cell), LipT-RvLacZ at 10⁵ lipids/pt yielded a 1.5-fold increase inreporter gene expression. LipT-RvLacZ at 10⁶ lipids/pt gave a 2.3-foldincrease in the level of expression as compared to RvLacZ only. Theliposome complex without transferrin gave only limited enhancement.Therefore, the optimal ratios of LipT-RvLacZ complex appeared to beabout 10-1000 cationic lipids/Tf molecule, and about 10⁴⁻¹⁰ ⁷ cationiclipids/pt, preferably about 15-50 cationic lipids/Tf molecule and about10⁶ cationic lipids/pt. If the lipid/pt ratio is too high, precipitationcan occur.

Histochemical staining shows that RvLacZ alone gave 20-30% transductionefficiency while transferrin-liposome complexed retrovirus LipT-RvLacZ(106 lipids/pt) gave 60-80% efficiency.

The above results demonstrated that transferrin-cationic liposomes cancomplex with retrovirus and substantially enhance retroviral genetransduction.

EXAMPLE 24 Electron Microscopic Analysis of Ligand-Liposome-DNA complex

Liposomes can be observed under an electron microscope (EM), such as atransmission electron microscope (TEM) with negative staining or ascanning electron microscope (SEM). EM can reveal the structure and sizedistribution of the liposome complexes. EM can also be used for qualitycontrol of liposomal preparation.

In this example, we demonstrate a new, unique transferrin-liposomalstructure, one that may account for the stability and efficacy observedwith the ligand-liposomal-therapeutic molecule of the inventiondescribed in this application.

We observed the ligand-cationic liposomes under Transmission ElectronMicroscope with negative staining. A copper grid with Formvar and Carboncoating (Electron Microscopy Sciences, Fort Washington, Pa.) was used inthe study. Ligand-liposome-pCMVp53 complexes were prepared as describedin Examples 2 and 17. One drop of liposome complex was placed on thegrid. After 5 minutes, excess liquid was removed by capillary actionwith filter paper at the edge of the grid. One drop of 4% UraniumAcetate was then added to the grid for negative staining. After 5minutes, excess liquid was also removed as above. The grid was air driedat room temperature for 15 min before being put into the sample chamberof TEM. The JOEL 1200EX or JOEL 100S were used in the study according tothe manufacturer's instruction. Photos were taken at magnitudes of 10-50k, 60 kVolt. The liposome samples on the grid were prepared, freshlystained and observed within one hour.

Many publications have indicated that cationic liposome-DNA complexeshave a diverse structure and size ranging from 100 nm to 1000 nm. In ourstudy, we observed unexpectedly that the ligand-liposome-DNA complexesprepared in accordance with this invention have much smaller size andmuch more even size distribution. In particular, LipT(A)-p53 complexeshave a size ranging from about 30-100 nm in diameter, preferably 35-65nm (averaging about 50 nm). As the cationic liposome Lip(A) itself has asize of 15-40 nm, averaging 25 nm, when transferrin was complexed withLip(A), the size did not change appreciably. However, thicker liposomalwalls or membranes were observed, indicating that transferrin wascomplexed onto the liposome membrane. From the enlarged photos weobserved an irregular or acentric onion-like structure in the core ofthe LipT(A)-DNA complex. An intermediate stage of formation of thestructure, e.g., an intermediate step in the condensation of the DNAchain by LipT(A), was observed as well. When the incubation time formixing LipT(A) with DNA was shortened from 15 to 5 minutes, more of thisintermediate stage was observed.

Based upon the TEM observations, it appears that the unique structure ofthe LipT-DNA complex may play in important role in the high genetransfection efficiency observed in vitro and especially in vivo. Theacentric onion-like core structure may be formed via the following stepsduring the formation of the LipT(A)-DNA complex:

Step 1. Several (4-8 or more) Tf-liposomes contact each DNA molecule,attaching to the DNA chain though electrostatic interaction.

Step 2. Each attached Tf-liposome wraps or condenses the DNA chain toform individual lamellar structures along the DNA chain.

Step 3. The lamellar structures condense to form one core lamellarstructure. This solid core structure is smaller in size than the sum of4-8 Tf-liposomes.

Step 4. During the final condensation, a phase transition from lamellarphase to an inverted hexagonal phase may occur, giving rise to theirregular or acentric onion-like structure.

The inverted hexagonal (H_(II)) phase is believed to be substantiallymore efficient than the lamellar (L_(II)) phase at transfection and maybe related to DNA release and delivery (Koltover, I. Science281:78.1998). Using freeze-fracture electron microscopy, Sternberg, B.(Biochim Biophys Acta 1998; 1375:23-35) described a “mappin” structurein DDAB/Chol cationic liposome-DNA complexes that had highest in vivotransfection activity. This high in vivo activity, he believed, isrelated to small (100-300 nm) stabilized complexes whereas high in vitroactivity is associated with hexagonal lipid precipitates. Noultrastructural analysis of ligand-cationic liposome-DNA complexes isavailable in literature. We believe that in the presence of transferrinor other ligands, the L_(II) to H_(II) transition tends to occur and theformed irregular or acentric onion-like core structure is stabilized bythe ligand. As for the mechanism of lamellar-to-inverted-hexagonal phasetransition, besides that suggested by Koltover, the ligands may play animportant role. Tf attached on liposomal surface or folate linked on theliposomal surface may help or accelerate the phase transition, givingrise to the highly efficient acentric onion-like core structures.

In the preparation conditions disclosed herein, more than 95% ofLipT(A)-DNA complexes have the irregular or acentric onion-like corestructure. If not for this transition, the condensed lamellar structuresin Step 3-4 will preferably form regular or centered onion-like corestructure to be stable. This L_(II) to H_(II) transition andTf-stabilization may account for the unexpectedly high in vivo genetransfection efficiency.

Since the complexation is a four-step process, it is important, whenpreparing the complex, to incubate for a sufficient period of timebetween each mixing step, using frequent shaking, to permit the acentriconion-like core structure to form completely. For the preparationprocedures disclosed herein, the incubation time should be about 5-15minutes after each mixing and about 10-30 minutes after mixing with DNA,preferably about 15-30 minutes.

Another unique feature of the liposomes according to the invention istheir evenly distributed smaller size (diameter less than about 100 nm,preferably less than about 75 nm, more preferably about 35-65 nm (50 nmaverage) diameter). To reach the target tumor in vivo, the liposomesmust first be resistant to serum and then pass through the blood vessel(capillary) wall. The complexes of the present invention exhibit highresistance to degredation by serum. The permeable size of thecapillaries in tumors is usually 50-75 nm; therefore, the complexes canpass through the capillary wall to reach the target.

The TEM structure of LipF(B)-DNA complex is similar to that ofLipT(A)-DNA, and this complex has a size range of 30-100 nm, preferably35-75 nm (average 50 nm) in diameter. The unique irregular or acentriconion-like core structures were also observed. Thelamellar-to-inverted-hexagonal phase transition may occur in a similar4-step process, which accounts for the unexpectedly high in vivo genetransfection efficiency.

EXAMPLE 25 Stability of the Ligand-cationic Liposomes

Stability is an important issue for liposomal pharmaceuticals. Liposomesolutions should be stable for an extended period of time afterpreparation to allow for shipment and storage without substantial lossof their biological/pharmaceutical activities, to be useful astherapeutic agents. In light of the future clinical use of theligand-liposome-therapeutic molecule complex of this invention, weexamined the stability of the ligand-liposomes and theligand-liposome-DNA complexes.

Lip(A) was prepared in water and stored under nitrogen in the dark at 4°C. for various periods of time, up to 6 months. On the day of the assay,the stored liposomes, as well as freshly prepared Lip(A), was used tomake the LipT(A)-pCMVb complex. The complex was then used to transfectJSQ-3 cells using the transfection assay as described in Example 5. Noappreciable difference in the level of the transgene expression wasobserved between the Lip(A) preparations which had been in storage forvarious lengths of time and the freshly prepared Lip(A). In a separateexperiment, a Lip(A) preparation stored for 12 months stillretained >90% of its transfection activity. The transferrin solution (5mg/ml in water) and pCMVb plasmid DNA (0.5-1.0 μg/ml) in 10 mM Tris-HCl,1 mM EDTA, pH 8.0) were each prepared separately. Folate-liposomecomplexes were found to have the same degree of stability.

The liposomes, Tf, and plasmid DNA are all individually stable instorage. But, when they are mixed together to form the LipT-DNA complex,the complex is unstable for an extended period of time. For example, theLipT-DNA complex was stable for only a few days. On day 3, only 50%transfection activity remained. For LipF-DNA, only 60% transfectionactivity remained after 24 hours, with virtually complete loss ofactivity 3 days after preparation.

Based upon these observations, it appears that the components of theligand-liposome-therapeutic molecule complexes of this invention canadvantageously be provided in kit form. The components can be mixedtogether sequentially, on the day of use, by first adding the Tf to theliposome, followed by the DNA solution (incubating 10-15 min. betweeneach mixing) then adding dextrose to 5%. The complex should beadministered as quickly as practical, preferably within 24 hours,following its preparation.

We claim:
 1. A vector for the systemic delivery of a diagnostic oranti-tumor agent to a target cell within a host animal, comprising acomplex of a cell-targeting ligand, a cationic liposome comprising acationic lipid selected from dioleoyltrimethylammonium-propane (DOTAP)or dimethyl dioctadecylammonium bromide (DDAB) and neutral or helperlipid dioleoylphosphatidylethanolamine (DOPE), and said diagnostic oranti-tumor agent, wherein the vector has a mean diameter of less thanabout 100 nm and the ligand is bound directly to the liposome.
 2. Thevector according to claim 1 having a mean diameter of about 30 to 75 nm.3. The vector according to claim 1 having a mean diameter of about 50nm.
 4. The vector according to claim 1 wherein said agent is a nucleicacid.
 5. The vector according to claim 1 wherein said agent encodes (a)a protein or a (b) an antisense oligonucleotide.
 6. The vector accordingto claim 1 wherein said agent is a nucleic acid encoding wild-type p53.7. The vector according to claim 1 wherein said ligand is a tumor celltargeting ligand.
 8. The vector according to claim 1 wherein said ligandis folate or transferrin.
 9. The vector according to claim 1 whereinsaid ligand is folate.
 10. The vector according to claim 1 wherein saidligand is transferrin.
 11. The vector according to claim 4 wherein saidliposome and said nucleic acid are present at a ratio ranging from0.1-50 nanomoles liposome per 1.0 μg nucleic acid.
 12. The vectoraccording to claim 11 wherein said ratio ranges from 1.0-24 nanomoleliposome per 1.0 μg nucleic acid.
 13. The vector according to claim 11wherein said ratio ranges from 6-16 nanomoles liposome per 1.0 μgnucleic acid.
 14. The vector according to claim 1 wherein said vectorhas an acentric structure.
 15. The vector according to claim 14 whereinsaid vector has a solid core.
 16. A vector for delivering in vivo atherapeutically effective nucleic acid molecule to a tumor-bearinganimal, the vector consisting essentially of a complex of acell-targeting ligand selected from the group consisting of folate andtransferrin, a cationic liposome comprising a cationic lipid selectedfrom DOTAP or DDAB and neutral or helper lipid DOPE, and a nucleic acidmolecule, wherein said vector has a mean diameter of less than about 100nm and the folate or transferrin ligand is bound directly to saidliposome.
 17. The vector of claim 16 wherein said nucleic acid moleculeencodes wild type p53.
 18. The vector of claim 16 wherein said liposomeand said nucleic acid molecule are in a ratio of 0.1-50 nanomoleliposome per 1.0 μg nucleic acid.
 19. The vector of claim 16 whereinsaid liposome and said nucleic acid molecule are in a ratio of 1.0-24nanomole liposome per 1.0 μg nucleic acid.
 20. The vector of claim 16wherein said liposome and said nucleic acid molecule are in a ratio of6-16 nanomole liposome per 1.0 μg nucleic acid.
 21. The vector of claim16 wherein said vector has an acentric structure.
 22. The vector ofclaim 21 wherein said vector has a solid core.
 23. A pharmaceuticalcomposition comprising a vector according to claim 16 in apharmaceutically acceptable carrier.
 24. A method for systemicallyproviding a therapeutic anti-tumor agent to an animal in need thereof,comprising systemically administering to said animal a therapeuticallyeffective amount of a complex comprising a cell-targeting ligand, acationic liposome comprising a cationic lipid selected from DOTAP orDDAB and neutral or helper lipid DOPE, and said therapeutic anti-tumoragent, wherein said vector has a mean diameter of less than about 100 nmand said ligand is bound directly to said liposome.
 25. The method ofclaim 24 wherein said agent is a nucleic acid.
 26. The method of claim25 wherein said liposome and said nucleic acid are present at a ratioranging from 0.1-50 nanomole liposome per 1.0 μg nucleic acid.
 27. Themethod of claim 25 wherein said liposome and said nucleic acid arepresent at a ratio ranging from 1-24 nanomole liposome per 1.0 μgnucleic acid.
 28. The method of claim 25 wherein said liposome and saidnucleic acid are present at a ratio ranging from 6-16 nanomole liposomeper 1.0 μg nucleic acid.
 29. The method of claim 24 wherein said complexhas an acentric structure.
 30. The method of claim 29 wherein saidcomplex has a solid core.
 31. The method according to claim 24, whereinsaid vector is administered intravenously.
 32. The method according toclaim 24, wherein the cell-targeting ligand is folate or transferrin,and the therapeutic anti-tumor agent is a nucleic acid encodingwild-type p53.
 33. The method according to claim 24 wherein the vectoris administered in a pharmaceutically acceptable composition comprisinga pharmaceutically acceptable vehicle.
 34. A therapeutic method for thetreatment or amelioration of cancer in a warm blooded animal, comprisingadministering to said animal a complex comprising a cancer celltargeting ligand, a cationic liposome comprising a cationic lipidselected from DOTAP or DDAB and neutral or helper lipid DOPE, and atherapeutic nucleic acid, wherein said complex has a mean diameter ofless than about 100 nm and the ligand is bound directly to the liposome.35. The method of claim 34 wherein said liposome and said nucleic acidare present at a ratio ranging from 0.1-50 nanomole liposome per 1.0 μgnucleic acid.
 36. The method of claim 35 wherein said liposome and saidnucleic acid are present at a ratio ranging from 1-24 nanomole liposomeper 1.0 μg nucleic acid.
 37. The method of claim 35 wherein saidliposome and said nucleic acid are present at a ratio ranging from 6-16nanomole liposome per 1.0 μg nucleic acid.
 38. The method of claim 34wherein said complex has an acentric structure.
 39. The method of claim38 wherein said complex has a solid core.
 40. The therapeutic methodaccording to claim 34 wherein said complex is comprised of acell-targeting ligand selected from the group consisting of folate andtransferrin, a cationic liposome and a nucleic acid encoding wild-typep53.
 41. The therapeutic method according to claim 40 wherein saidcomplex is systemically administered to a cancer-bearing warm bloodedanimal.
 42. The therapeutic method according to claim 40, wherein saidcomplex is intravenously administered to a cancer-bearing warm bloodedanimal.
 43. The therapeutic method according to claim 40, wherein saidcomplex is intratumorally administered to a cancer-bearing warm bloodedanimal.
 44. The therapeutic method according to claim 40, furthercomprising administering an anti-cancer chemotherapeutic agent or ananti-cancer radiotherapy to said animal.
 45. A method for preparingcomplexes smaller than 100 nm in diameter wherein said complexescomprise a cationic liposome comprising a cationic lipid and a neutralor helper lipid, a ligand and a nucleic acid, said method comprising thesteps of: a) mixing said ligand with said cationic liposome to form acationic liposome:ligand complex; and b) mixing said cationicliposome:ligand complex and said nucleic acid at a ratio of from 0.1-50nanomoles liposome per 1.0 μg nucleic acid to form a cationicliposome:ligand:nucleic acid complex; wherein said cationic lipidcomprises dioleoyltrimethylammonium-propane (DOTAP) or dimethyldioctadecylammonium bromide (DDAB) and said neutral or helper lipidcomprises dioleoylphosphatidylethanolamine (DOPE).
 46. The method ofclaim 45 wherein said ratio is from 1-24 nanomoles liposome per 1.0 μgnucleic acid.
 47. The method of claim 45 wherein said ratio is from 6-16nanomoles liposome per 1.0 μg nucleic acid.
 48. The method of claim 45wherein said ligand is folate or transferrin.
 49. The method of claim 45wherein said liposome:ligand complex of step (a) is incubated for 5-15minutes before performing step (b).
 50. The method of claim 34, whereinsaid cancer comprises breast cancer, prostate cancer, head and neckcancer, ovarian cancer, pancreatic cancer, colon cancer, glioblastoma,cervical cancer, lung cancer, gastric cancer, liposarcoma, melanoma orchoriocarcinoma.
 51. The method of claim 34, wherein said cancercomprises breast cancer, prostate cancer, head and neck cancer, orpancreatic cancer.
 52. The method of claim 51, wherein said ligandcomprises transferrin or folate and said therapeutic nucleic acidencodes wt p53.
 53. The method of claim 45, which further comprisescombining said complex with an aqueous solution of sucrose or dextrose.54. The vector of claim 1, wherein said cationic lipid and said neutralor helper lipid are present at a ratio of 1:(0.5-3) (molar ratio). 55.The vector of claim 10, wherein said nucleic acid, lipids and ligand arepresent in a ratio of 1 μg:(0.1-50 nmol):(0.1-100 μg).
 56. The vector ofclaim 10, wherein said nucleic acid, lipids and ligand are present in aratio of 1 μg:(5-24 nmol):(6-36 μg).